RU2756275C2 - Antibodies specific to human poliovirus receptor (pvr) - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology, in particular to a selected monoclonal antibody, which specifically binds to human poliovirus receptor (PVR), or to its fragment, as well as to a composition containing it. Polynucleotide encoding a sequence of variable area of light or heavy chain of the specified antibody, as well as a plasmid and a cell containing it are also disclosed.EFFECT: invention allows for effective diagnostics or treatment of cancer in a subject.31 cl, 16 dwg, 4 tbl, 13 ex

Description

The technical field to which the present invention relates

The present invention relates to the field of immunotherapy and relates to antibodies and fragments thereof that bind to a poliovirus receptor protein, polynucleotide sequences encoding such antibodies, and hybridoma cells producing such antibodies. The present invention further relates to therapeutic and diagnostic compositions containing such antibodies, and to methods of treating and diagnosing diseases, in particular cancer, using such monoclonal antibodies.

Prior art of the present invention

Cancer immunotherapy is used to generate and enhance an anti-tumor immune response, for example, by treatment with antibodies specific for antigens on the surface of tumor cells, using antigen-presenting cell-tumor cell hybrids, or by specifically activating anti-tumor T cells. The ability to recruit immune cells (eg, T cells) to tumor cells in a patient provides a therapeutic effect against cancers and metastasis types that have hitherto been considered incurable.

The T-cell mediated immune response includes many sequential stages, regulated by a certain ratio of costimulatory and co-inhibiting signals, which control the intensity of the immune response. Inhibitory signals, called immune checkpoints, are key to maintaining self-tolerance as well as limiting immune-mediated concomitant tissue damage. These signals undergo changes as immune clearance, aggravation or persistence of infection, and such changes affect the response of T cells and cause a change in the shape of the immune response.

The expression of proteins that are checkpoints of the immune response can be regulated by tumors. For example, up-regulation of programmed cell death protein-1 ligand (PD-L1) on the surface of cancer cells allows them to evade the host's immune system by inhibiting T cells by binding to PD-1, which would otherwise attack such tumor cells. Thus, checkpoints of the immune response are significant obstacles to the activation of functional cellular immunity against cancer. Accordingly, antagonist antibodies specific to inhibitory ligands on the surface of immune cells are considered promising anti-cancer agents and are being evaluated in clinical settings (eg nivolumab and pembrolizumab). Another example of a molecule that is a checkpoint of the immune response is the T-cell immunoreceptor with domains Ig and ITIM (TIGIT). TIGIT is a co-inhibiting molecule expressed on the surface of a number of immune cells, including T cells and natural killer (NK) cells. TIGIT binds with high affinity to the poliovirus receptor (PVR).

The poliovirus receptor (PVR), also called CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. It has previously been described as a tumor antigen and as a potential therapeutic target because its level of expression is increased in neuroectodermal malignancies, including glioblastoma multiforme, medulloblastoma, and colon and rectal carcinoma (Solecki et al., J. Biol. Chem. 2002, 277: 25697-700), as well as in pancreatic cancer (Nishiwada et al., Anticancer Res. 2015, 35 (4): 2287-97). It is also known that PVR enhances serum-induced activation of the Ras-Raf-MEK-ERK signaling pathway, upregulates cyclins D2 and E and downregulates p27Kip1, ultimately shortening the G0 / G1 phase of the cell cycle (Kakunaga 2004, J. Biological Chemistry , 279, 36419-36425), therefore, it is assumed that blocking PVR on the surface of tumor cells reduces the viability of tumor cells. PVR also plays an important role in the process of angiogenesis and is believed to mediate the regulation of VEGF-induced angiogenesis by controlling the interaction of VEGFR2 with integrin α (v) β (3) and the VEGFR2-mediated Rap1-Akt signaling pathway (Kinugasa et al., 2012, Circ Res. 2012,110 (5), 716-26). In addition, PVR forms a complex with IGF1R and is involved in Met signaling, and blocking complex formation reduced cell viability and angiogenesis (Lee et al., Scientific Reports 2014, 20, 4, 7139).

The involvement of PVR in the metastasis process has been demonstrated by injecting cancer cells into the tail of mice and monitoring the extent of lung metastasis. It has been shown that PVR, which is upregulated in cancer cells, cross-interacts with its counter-receptor on the platelet surface, and that this cross-interaction enhances metastasis of cancer cells to the lungs (Morimoto et al., Oncogene (2008) 27, 264-273) ...

US Patent Application Publication No. 20070041985 discloses molecules that specifically bind to at least one intra- or extracellular domain of PVR or any derivative thereof, wherein the molecule is characterized by the ability to modulate the pattern of receptor-mediated adhesion, migration and / or invasion of a cell expressing PVR or any its derivative.

US Patent Application Publication No. 20090215175 discloses molecules (eg, low molecular weight chemicals, oligonucleotides, polypeptides, antibodies, and antibody fragments) that modulate PVR functions required to promote cell adhesion, migration, invasion, and / or metastatic potential. These molecules can be used to treat cells with metastatic potential, metastasis and cancer.

PCT Application Publication No. WO 2006/124667 discloses the modulation of the zB7R1 protein (TIGIT) with monoclonal antibodies that block the binding of TIGIT to its PVR ligand.

There is an unmet need to provide additional and more effective, specific, safe and / or stable agents that, alone or in combination with other agents, stimulate cells of the immune system to attack tumor or virus-infected cells by inhibiting the binding of PVR to TIGIT.

Brief disclosure of the present invention

The present invention relates to antibodies and fragments thereof that recognize the poliovirus receptor (PVR), prevent it from binding to the T-cell immunoreceptor with the Ig and ITIM domains (TIGIT) and inhibit the suppressor activity on lymphocytes such as natural killer (NK) cells and T cells. The anti-PVR antibodies disclosed herein are capable of binding to PVR present on the surface of cancer cells. Such antibodies and their fragment are characterized by the presence of unique sets of complementarity determining regions (CDRs) sequences, high affinity and high specificity for PVR, and are useful in cancer immunotherapy to counteract tumor evasion from immune surveillance, as a separate therapy and in combination with other anti-cancer agents. ... Antibodies are also useful in the treatment of viral infections.

It is disclosed below that the high affinity anti-PVR antibodies disclosed herein block the TIGIT-PVR interaction and reactivate T and NK cells. The antibodies have been shown to be highly specific for human PVR. These properties make the monoclonal antibodies (mAbs) of the present invention valuable candidates for use in cancer immunotherapy, allowing lower doses to be administered with fewer side effects.

Preferably, the anti-PVR mAb of the present invention can induce T cell proliferation to a greater extent than the anti-PD-1 mAb and CTLA-4 mAb in an in-vitro model using PD-L1 (A549). An inductive effect was observed for peripheral blood mononuclear cells (PMBCs) and purified CD4 and CD8 T cells. In addition, anti-PVR mAbs were able to induce NK cell activation among the majority of target cells tested. Moreover, some of the anti-PVR antibodies described herein exhibited in-vitro anti-cancer activity comparable to that of the known agent, Erbitux®, which is currently used in therapy. In addition, some of the anti-PVR antibodies described herein have shown a synergistic effect when compared with additional anti-cancer agents such as anti-PD-1 and CTLA-1 antibodies and epidermal growth factor receptor (EGFR). In addition, some of the anti-PVR antibodies have been found to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Additionally, it is disclosed that some anti-PVR antibodies in accordance with the present invention do not have a blocking effect on costimulatory signaling by DNAM1, therefore, they do not have a deleterious effect on other signals that mediate the induction of an immune response.

Interestingly, despite the high degree of sequence similarity among human and rodent PVR sequences, the antibodies of the present invention are highly specific for human PVR.

Additionally, it is disclosed that, surprisingly, certain chimeric monoclonal antibodies containing a human constant chain have shown an enhanced effect on the activation of immune cells compared to their equivalent mouse monoclonal antibodies.

Several of the anti-PVR mAbs described herein were able to reduce the viability of tumor cells in an immune-independent manner by blocking PVR on the surface of tumor cells. Without going into any mechanism of action, it is believed that such activity is a result of the ability of PVR to shorten the duration of the G0 / G1 phase of the cell cycle.

In one aspect, the present invention provides an isolated monoclonal antibody that binds to a poliovirus receptor (PVR), or an antibody fragment thereof, comprising at least an antigen binding region, wherein the isolated antibody or antibody fragment is selected from the group consisting of:

i. three CDRs of the variable region of the heavy chain (HC) containing SEQ ID NO: 77, and three CDRs of the variable region of the light chain (LC) containing SEQ ID NO: 79, or an analog or derivative thereof, characterized by at least 90% sequence identity with the specified sequence of the antibody or fragment;

ii. three complementarity determining regions (CDRs) of the variable region of the heavy chain containing SEQ ID NO: 69, and three CDRs of the variable region of the light chain containing SEQ ID NO: 71, or their analog or derivative, characterized by at least 90% sequence identity with the specified an antibody or fragment sequence; and

iii. three CDRs of the variable region of the heavy chain, containing SEQ ID NO: 73, and three CDRs of the variable region of the light chain, containing SEQ ID NO: 75, or an analog or derivative thereof, characterized by at least 90% sequence identity with the specified sequence of the antibody or fragment.

Antibodies containing CDR sequences contained in heavy and light chains homologous to SEQ ID No: 2, 10, 18, 26, 34 or 42 are also included within the scope of the present invention. In some embodiments, SEQ ID NO: 2 is interchangeable with SEQ ID NO: 69. In some embodiments, SEQ ID NO: 10 is interchangeable with SEQ ID NO: 71. In some embodiments, SEQ ID NO: 18 is interchangeable with SEQ ID NO: 73. In some embodiments, SEQ ID NO: 26 is interchangeable with SEQ ID NO: 75. In some embodiments, SEQ ID NO: 34 is interchangeable with SEQ ID NO: 77. In some embodiments, SEQ ID NO: 42 is interchangeable with SEQ ID NO: 79.

There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard method that gives definite results. Determination of CDR sequences from the variable regions of the heavy and light chains of an antibody can be performed according to any method known in the art, including, without limitation, methods known as KABAT, Chothia, and IMGT. A selected set of CDRs can include sequences identified using more than one method, ie, some CDR sequences can be determined using KABAT, and some can be determined using, for example, IMGT.

In some embodiments, the isolated monoclonal antibody or fragment comprises the CDR sequences of the anti-PVR (or hPVR.07) monoclonal antibody designated 4E5, i.e., the three CDR sequences contained within the heavy chain variable region set forth in SEQ ID NO : 69, and three CDR sequences contained within the light chain variable region set forth in SEQ ID NO: 71.

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 comprising the sequence GFDFSRYW (SEQ ID NO: 4). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 comprising the sequence EIHPDSSKINYTPSQ (SEQ ID NO: 6). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR3 comprising the sequence PDGNYNALDYW (SEQ ID NO: 8).

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 containing the RYW sequence. In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 comprising the sequence RYWMT (SEQ ID NO: 80). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR3 comprising the sequence PDGNYNALDY (SEQ ID NO: 82).

In accordance with certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) an HC CDR1 comprising the sequence GFDFSRYW (SEQ ID NO: 4); (ii) CDR2 HC containing the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6); and (iii) CDR3 HC containing the sequence: PDGNYNALDYW (SEQ ID NO: 8).

In accordance with certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) an HC CDR1 comprising an RYW sequence; (ii) CDR2 HC containing the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6); and (iii) CDR3 HC containing the sequence: PDGNYNALDY (SEQ ID NO: 82).

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises light chain CDR1 comprising the sequence KASQDVGTAVT (SEQ ID NO: 12). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises light chain CDR2 containing the sequence WASTRHT (SEQ ID NO: 14). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 comprising the sequence QQYSRYPYT (SEQ ID NO: 16). In accordance with certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) an LC CDR1 comprising the sequence KASQDVGTAVT (SEQ ID NO: 12); (ii) CDR2 LC containing the sequence: WASTRHT (SEQ ID NO: 14); and (iii) CDR3 HC containing the sequence: QQYSRYPYT (SEQ ID NO: 16).

In accordance with some specific embodiments, the isolated monoclonal antibody or fragment comprises a heavy chain CDR1 sequence comprising the sequence: GFDFSRYW (SEQ ID NO: 4), a heavy chain CDR2 comprising the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3, containing the sequence: PDGNYNALDYW (SEQ ID NO: 8), light chain CDR1 containing the sequence: KASQDVGTAVT (SEQ ID NO: 12), light chain CDR2 containing the sequence: WASTRHT (SEQ ID NO: 14), and light chain CDR3 containing sequence: QQYSRYPYT (SEQ ID NO: 16), or their analogs, providing not more than 5% amino acid substitutions, deletions and / or insertions in the hypervariable region (HVR) sequence.

In some specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of:

i. Heavy chain CDR1 with a sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 80,

ii. Heavy chain CDR2 with a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 81,

iii. Heavy chain CDR3 with a sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 82,

iv. CDR1 light chain with the sequence set out in SEQ ID NO: 12,

v. Light chain CDR2 of the sequence set forth in SEQ ID NO: 14, and

vi. Light chain CDR3 with the sequence set out in SEQ ID NO: 16.

In accordance with some specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

In accordance with other specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

In some embodiments, an isolated monoclonal antibody or fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO: 69), or an analogue or derivative thereof, having at least 90% sequence identity with that of the heavy chain variable region.

In some embodiments, the SEQ ID NO: 2 analog is a heavy chain variable region of the sequence set forth in SEQ ID NO: 69.

In some embodiments, an isolated monoclonal antibody or fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 71), or an analogue thereof, having at least 90% sequence identity with a light chain variable region sequence.

In some embodiments, the SEQ ID NO: 10 analog is a light chain variable region of the sequence set forth in SEQ ID NO: 71.

In accordance with a specific embodiment, an isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region of the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 69 and a light chain variable region of the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 71, or an analogue thereof, having at least 90% sequence identity to the sequence of the light and / or heavy chains.

The present invention also encompasses an antibody or antibody fragment capable of binding with high affinity to an epitope within the human PVR protein to which the 4E5 monoclonal antibody (mAb) binds.

In other embodiments, the isolated monoclonal antibody comprises the CDR sequences of the monoclonal antibody designated 7D4 (or hPVR.01), i.e., the three CDR sequences contained within the heavy chain variable region set forth in SEQ ID NO: 73, and three CDR sequences contained within the light chain variable region set forth in SEQ ID NO: 75.

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 comprising the sequence GYTFTEYTMH (SEQ ID NO: 20). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 comprising the sequence GIDPNNGGTNYNQNFKG (SEQ ID NO: 22). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR3 containing the VIPLEY sequence (SEQ ID NO: 24). In accordance with certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) an HC CDR1 comprising the sequence GYTFTEYTMH (SEQ ID NO: 20); (ii) CDR2 HC containing the sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); and (iii) CDR3 HC containing the sequence: VIPLEY (SEQ ID NO: 24).

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 comprising the sequence EYTMH (SEQ ID NO: 83).

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises light chain CDR1 comprising the sequence KASQNVYTNVA (SEQ ID NO: 28). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 comprising the sequence SASYRYR (SEQ ID NO: 30). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises light chain CDR3 comprising the sequence QQYNSYPLA (SEQ ID NO: 32). In accordance with certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) an LC CDR1 comprising the sequence KASQNVYTNVA (SEQ ID NO: 28); (ii) CDR2 LC containing the sequence: SASYRYR (SEQ ID NO: 30); and (iii) CDR3 HC containing the sequence: QQYNSYPLA (SEQ ID NO: 32).

In some specific embodiments, an isolated monoclonal antibody comprises a heavy chain CDR1 comprising the GYTFTEYTMH sequence (SEQ ID NO: 20), a heavy chain CDR2 comprising: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), a heavy chain CDR3 comprising the sequence: VIPLEY (SEQ ID NO: 24), light chain CDR1 containing the sequence: KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 containing the sequence: SASYRYR (SEQ ID NO: 30), and light chain CDR3 containing the sequence: QQYNSYPLA ( SEQ ID NO: 32), or their analogs, providing not more than 5% of amino acid substitutions, deletions and / or insertions in the HVR sequence.

In accordance with some specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: heavy chain CDR1 with a sequence selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 83, heavy chain CDR2 with the sequence of SEQ ID NO: 22, the heavy chain CDR3 of the sequence of SEQ ID NO: 24, the light chain CDR1 of the sequence of SEQ ID NO: 28, the light chain CDR2 of the sequence of SEQ ID NO: 30, and the light chain CDR3 of the sequence set forth in SEQ ID NO: 32.

In accordance with some specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 32.

In accordance with other specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 83, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 32.

In some embodiments, an isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 73, or an analogue or derivative thereof, having at least 90% sequence identity with a heavy chain variable region sequence.

In some embodiments, the SEQ ID NO: 18 analog is a heavy chain variable region of the sequence set forth in SEQ ID NO: 73.

In some embodiments, an isolated monoclonal antibody or fragment thereof comprises the light chain variable region set forth in SEQ ID NO: 75, or an analogue thereof, having at least 90% sequence identity with that of the light chain variable region.

In some embodiments, the SEQ ID NO: 26 analog is a light chain variable region characterized by the sequence set forth in SEQ ID NO: 75

In accordance with a particular embodiment, an isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region of the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 73 and a light chain variable region of the sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 75, or an analogue thereof, having at least 90% sequence identity to the sequence of the light and / or heavy chains.

The present invention also encompasses an antibody or antibody fragment capable of binding with high affinity to an epitope within the human PVR protein to which mAb 7D4 binds.

In other embodiments, the isolated monoclonal antibody comprises the CDR sequences of the monoclonal antibody designated 5B9 (or hPVR.09), i.e., three CDR sequences contained within the heavy chain variable region set forth in SEQ ID NO: 77, and three CDR sequences contained within the light chain variable region set forth in SEQ ID NO: 79.

In accordance with some embodiments, an isolated monoclonal antibody comprises complementarity determining region (CDR) sequences contained within a heavy chain variable region set forth in SEQ ID NO: 34 and three CDR sequences contained within a light chain variable region sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54. Each variant is a separate embodiment of the present invention.

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 comprising the sequence GYTFSNYWIE (SEQ ID NO: 36). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 comprising the sequence EIFPGSGRINFNEKFKG (SEQ ID NO: 38). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR3 comprising the sequence TKIYGNSFDY (SEQ ID NO: 40). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises heavy chain CDR1 containing the sequence SNYWIE (SEQ ID NO: 84).

In accordance with certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) an HC CDR1 comprising the sequence SNYWIE (SEQ ID NO: 84); (ii) CDR2 HC containing the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38); and (iii) CDR3 HC containing the sequence: TKIYGNSFDY (SEQ ID NO: 40).

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises light chain CDR1 comprising the sequence KASQDVGTAVV (SEQ ID NO: 44). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises light chain CDR2 containing the sequence WASSRHN (SEQ ID NO: 46). In some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 comprising the sequence QQYSRYPLT (SEQ ID NO: 48).

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises light chain CDR1 comprising the sequence KASQDVGTAV (SEQ ID NO: 85).

In accordance with certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) an LC CDR1 comprising the sequence KASQDVGTAV (SEQ ID NO: 85); (ii) CDR2 LC containing the sequence: WASSRHN (SEQ ID NO: 46); and (iii) CDR3 HC containing the sequence: QQYSRYPLT (SEQ ID NO: 48).

According to additional embodiments, the LC CDR2 comprises the sequence set forth in SEQ ID NO: 46, 56, 57, 58, 59, 60, or 61. Each is a separate embodiment of the present invention.

In some specific embodiments, the isolated monoclonal antibody or fragment comprises a heavy chain CDR1 sequence comprising the sequence: GYTFSNYWIE (SEQ ID NO: 36), a heavy chain CDR2 comprising the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3, containing the sequence: TKIYGNSFDY (SEQ ID NO: 40), light chain CDR1 containing the sequence: KASQDVGTAVV (SEQ ID NO: 44), light chain CDR2 containing the sequence: WASSRHN (SEQ ID NO: 46), and light chain CDR3 containing sequence: QQYSRYPLT (SEQ ID NO: 48), or their analogs, providing not more than 5% of amino acid substitutions, deletions and / or insertions in the HVR sequence.

In accordance with some specific embodiments, the isolated monoclonal antibody or fragment consists of: heavy chain CDR1 with a sequence selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 84, heavy chain CDR2 with the sequence set forth in SEQ ID NO : 38, heavy chain CDR3 with the sequence set forth in SEQ ID NO: 40, light chain CDR1 with a sequence selected from the group consisting of SEQ ID NO: 44 and SEQ ID NO: 85, light chain CDR2 with the sequence set forth in SEQ ID NO: 46, and the light chain CDR3 of the sequence set forth in SEQ ID NO: 48.

In some specific embodiments, the isolated monoclonal antibody or fragment consists of: heavy chain CDR1 of SEQ ID NO: 36 or SEQ ID NO: 84, heavy chain CDR2 of SEQ ID NO: 38, CDR3 heavy chain with the sequence set forth in SEQ ID NO: 40, light chain CDR1 with the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 85, light chain CDR2 with a sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61; and light chain CDR3 of the sequence set forth in SEQ ID NO: 48. Each variant is a separate embodiment of the present invention.

In some embodiments, an isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 77, or an analogue or derivative thereof, having at least 90% sequence identity with a heavy chain variable region sequence.

In some embodiments, the SEQ ID NO: 34 analog is a heavy chain variable region of the sequence set forth in SEQ ID NO: 77.

In some embodiments, an isolated monoclonal antibody or fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 79, or an analogue thereof, having at least 90% sequence identity with a light chain variable region sequence.

In some embodiments, the SEQ ID NO: 42 analogue is a light chain variable region characterized by the sequence set forth in SEQ ID NO: 79.

In accordance with a specific embodiment, an isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region with a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 77, and a light chain variable region with a sequence selected from the group, consisting of SEQ ID NO: 42 and SEQ ID NO: 79, or their analogue, characterized by at least 90% sequence identity with the sequence of the light and / or heavy chains.

In some embodiments, an isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region of the sequence set forth in SEQ ID NO: 34 and a light chain variable region set forth in SEQ ID NO: 42, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54; or their analog, characterized by at least 90% sequence identity with the sequence of the light and / or heavy chains. Each embodiment is a separate embodiment of the present invention.

The present invention also encompasses an antibody or antibody fragment capable of binding with high affinity to an epitope within the human PVR protein to which mAb 5B9 binds.

In some embodiments, the isolated antibody or fragment thereof recognizes human PVR with an affinity of at least 10 ^-8 M. In other embodiments, the antibody or antibody fragment binds to human PVR with an affinity of 10 ^-8 M, 5 x 10 ^-9 M, 10 ^-9 M, 5 × 10 ^-10 M, 10 ^-10 M, 5 × 10 ^-11 M or even higher. Each embodiment is a separate embodiment of the present invention.

Analogs and derivatives of the isolated mAb antibodies and antibody fragments described above are also within the scope of the present invention. In some embodiments, particular analogs or isolated mAbs, or a fragment thereof, comprising at least one variable region represented by a sequence selected from the group consisting of: SEQ ID NOs: 2, 10, 18, 26, 34, and 42 are also included in the scope of the present invention. Isolated mAbs or a fragment thereof containing at least one variable region represented by a sequence selected from the group consisting of: SEQ ID NOs: 69, 71, 73, 75, 77 and 79 are also within the scope of the present invention.

In some embodiments, the antibody analog or antibody fragment has at least 90% sequence identity to the hypervariable region of the reference antibody sequence.

In accordance with certain embodiments, the analog or derivative of an isolated antibody or fragment thereof has at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the variable region of the reference antibody sequence. Each embodiment is a separate embodiment of the present invention.

In some embodiments, an antibody or antibody fragment of the present invention comprises a heavy chain variable region set forth in SEQ ID NO: 2 or SEQ ID NO: 69, or an analogue having at least 95% sequence similarity to said sequence. In other embodiments, an antibody or antibody fragment of the present invention comprises the variable region of the heavy chain set forth in SEQ ID NO: 18 or SEQ ID NO: 73, or an analogue having at least 95% sequence similarity to said sequence. In other embodiments, an antibody or antibody fragment of the present invention comprises the variable region of the heavy chain set forth in SEQ ID NO: 34 or SEQ ID NO: 77, or an analogue having at least 95% sequence similarity to said sequence.

In some embodiments, the antibody or antibody fragment comprises the light chain variable region set forth in SEQ ID NO: 10 or SEQ ID NO: 71, or an analogue having at least 95% sequence similarity to said sequence. In other embodiments, the antibody or antibody fragment comprises the light chain variable region set forth in SEQ ID NO: 26 or SEQ ID NO: 75, or an analogue having at least 95% sequence similarity to said sequence. In other embodiments, the antibody or antibody fragment comprises the light chain variable region set forth in SEQ ID NO: 42 or SEQ ID NO: 79, or an analogue having at least 95% sequence similarity to said sequence.

In some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, where: (i) the heavy chain comprises SEQ ID NO: 2 and the light chain comprises SEQ ID NO: 10; (ii) the heavy chain contains SEQ ID NO: 18 and the light chain contains SEQ ID NO: 26; or (iii) the heavy chain contains SEQ ID NO: 34 and the light chain contains SEQ ID NO: 42. Analogs of antibodies or fragments having at least 95% sequence similarity to said heavy or light chains are also included.

In other embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, wherein: (i) the heavy chain comprises SEQ ID NO: 69 and the light chain comprises SEQ ID NO: 71; (ii) the heavy chain contains SEQ ID NO: 73 and the light chain contains SEQ ID NO: 75; or (iii) the heavy chain contains SEQ ID NO: 77 and the light chain contains SEQ ID NO: 79. Analogs of antibodies or fragments having at least 95% sequence similarity to said heavy or light chains are also included.

In some embodiments, the analog has at least 96, 97, 98, or 99% sequence identity to the antibody light or heavy chain variable regions described above. In some embodiments, the analog provides no more than one amino acid substitution, deletion, or addition in one or more CDR sequences of the hypervariable region, namely, any of the CDR sequences set forth in SEQ ID NO: 4, 6, 8, 12, 14, 16, 20, 22, 24, 28, 30, 32, 36, 38, 40, 44, 46, 48, 80, 81, 82, 83, 84 and 85. Each embodiment is a separate embodiment of the present invention. In some embodiments, the amino acid substitution is a conservative substitution.

In some embodiments, the antibody or antibody fragment comprises a hypervariable region (HVR) with light and heavy chain regions as defined above in which 1, 2, 3, 4, or 5 amino acids have been substituted, removed and / or added. Each embodiment is a separate embodiment of the present invention.

In some embodiments, the antibody or antibody fragment comprises HVRs with light and heavy chain regions as defined above in which one amino acid has been replaced. In certain embodiments, the antibody or antibody fragment comprises a CDR as defined above in which one amino acid has been replaced. In some specific embodiments, the antibody or antibody fragment comprises a light chain CDR2 as defined above in which one amino acid has been replaced.

In some specific embodiments, the antibody or antibody fragment comprises light chain CDR2s of the sequence set forth in SEQ ID NO: 55 (WASSRHX), wherein X is selected from the group consisting of A, R, D, E, P, and T. Each variant is a separate embodiment of the present invention.

In some embodiments, the antibody or antibody fragment comprises a light chain CDR2 with a sequence selected from the group consisting of SEQ ID NO: 56-61.

In some embodiments, the isolated monoclonal antibody or antibody fragment comprises a set of CDRs selected from the group consisting of:

i. a CDR set of six CDRs, where: CDR1 HC is selected from GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84); CDR2 HC is EIFPGSGRINFNEKFKG (SEQ ID NO: 38); CDR3 HC is TKIYGNSFDY (SEQ ID NO: 40); CDR1 LC selected from KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85); CDR2 LC is selected from the group consisting of: WASSRHN (SEQ ID NO: 46), WASSRHA (SEQ ID NO: 56), WASSRHR (SEQ ID NO: 57), WASSRHD (SEQ ID NO: 58), WASSRHE (SEQ ID NO : 59), WASSRHP (SEQ ID NO: 60) and WASSRHT (SEQ ID NO: 61); and

CDR3 LC is QQYSRYPLT (SEQ ID NO: 48).

ii. a CDR set of six CDRs, where: the HC CDR1 sequence is selected from GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); CDR2 HC is selected from EIHPDSSKINYTPSQ (SEQ ID NO: 6) and EIHPDSSKINYTPSQKD (SEQ ID NO: 81); CDR3 HC is selected from PDGNYNALDYW (SEQ ID NO: 8) and PDGNYNALDY (SEQ ID NO: 82); CDR1 LC is KASQDVGTAVT (SEQ ID NO: 12); CDR2 LC is WASTRHT (SEQ ID NO: 14); and CDR3 LC is QQYSRYPYT (SEQ ID NO: 16).

iii. a CDR set of six CDRs, where: the HC CDR1 sequence is selected from GYTFTEYTMH (SEQ ID NO: 20) and EYTMH (SEQ ID NO: 83); CDR2 HC is GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); CDR3 HC is VIPLEY (SEQ ID NO: 24); CDR1 LC is KASQNVYTNVA (SEQ ID NO: 28); CDR2 LC is SASYRYR (SEQ ID NO: 30); and CDR3 LC is QQYNSYPLA (SEQ ID NO: 32).

Thus, the present invention relates to a monoclonal antibody that specifically binds a human PVR protein, or a binding fragment thereof, wherein said monoclonal antibody or fragment comprises a set of six CDR sequences, wherein the set is selected from the group consisting of:

i. SEQ ID NO. 4, 6, 8, 12, 14 and 16;

ii. SEQ ID NO. 20, 22, 24, 28, 30 and 32;

iii. SEQ ID NO. 36, 38, 40, 44, 46 and 48;

iv. SEQ ID NO. 36, 38, 40, 44, 55 and 48;

v. SEQ ID NO. 80, 81, 82, 12, 14 and 16;

vi. SEQ ID NO. 83, 22, 24, 28, 30 and 32;

vii. SEQ ID NO. 84, 38, 40, 85, 46 and 48; and

viii. SEQ ID NO. 84, 38, 40, 85, 55 and 48.

The present invention also relates to monoclonal antibodies and their binding fragments containing a heavy chain and a light chain, wherein said chains comprise a set of a heavy chain variable region sequence and a light chain variable region sequence, said set being selected from the group consisting of:

i. SEQ ID NO: 2 and 10;

ii. SEQ ID NO: 69 and 71;

iii. SEQ ID NO: 18 and 26;

iv. SEQ ID NO: 73 and 75;

v. SEQ ID NO: 34 and 42; and

vi. SEQ ID NO: 77 and 79.

In some embodiments, the antibody or antibody fragment is capable of inhibiting the binding of human PVR to TIGIT expressed on the surface of T cells or NK cells.

In accordance with a specific embodiment, the mAb is selected from the group consisting of: a chimeric antibody and an antibody fragment containing at least an antigen-binding portion of the antibody. In certain embodiments, the antibody is a chimeric antibody. In accordance with still other embodiments, the chimeric antibody comprises a human constant region. In accordance with still other embodiments, the chimeric monoclonal antibody comprises a human IgG1 constant region. In accordance with a specific embodiment, the antibody fragment is selected from the group consisting of: Fab, Fab ', F (ab') ₂ , Fd, Fd ', Fv, dAb, isolated CDR region, single chain antibody (scab), "diabody" and "linear antibodies". Each embodiment is a separate embodiment of the present invention.

In some embodiments, the antibody or antibody fragment comprises a framework sequence selected from the group consisting of: mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2, human IgG3, and human IgG4. Each embodiment is a separate embodiment of the present invention.

In some embodiments, a conjugate comprising an antibody or fragment thereof is provided as described above.

In some embodiments, the conjugate comprises a carrier protein.

In accordance with another aspect of the present invention, there are provided polynucleotide sequences encoding monoclonal antibodies characterized by high affinity and specificity for PVR, as well as vectors and host cells carrying such polynucleotide sequences.

In accordance with some embodiments, polynucleotide sequences encoding the HC variable region and light chain variable region (LC) amino acid sequences described above are provided.

In some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain capable of binding to an epitope within the human PVR protein to which binds: (i) a monoclonal antibody (herein identified as 4E5) with a heavy chain variable region SEQ ID NO: 2 and the light chain variable region of SEQ ID NO: 10; (ii) a monoclonal antibody (herein identified as 7D4) with a heavy chain variable region of SEQ ID NO: 18 and a light chain variable region of SEQ ID NO: 26; or (iii) a monoclonal antibody (herein identified as 5B9) with a heavy chain variable region of SEQ ID NO: 34 and a light chain variable region of SEQ ID NO: 42.

In some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 69. In some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 71.

In other embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 73. In additional embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 75.

In other embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 77. In additional embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 79.

In accordance with still some embodiments, the polynucleotide sequence of the present invention encodes an antibody or antibody fragment or chain comprising six CDR sequences: (i) heavy chain CDR1 with the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO : 80), heavy chain CDR2 with sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3 with sequence: PDGNYNALDY (SEQ ID NO: 82), light chain CDR1 with sequence: KASQDVGTAVT (SEQ ID NO: 12), CDR2 light chain with sequence: WASTRHT (SEQ ID NO: 14) and CDR3 light chain with sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chain CDR1 with sequence EYTMH (SEQ ID NO: 83), heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), light chain CDR1 with sequence: KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30) and light chain CDR3 with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) heavy chain CDR1 with sequence SNYWIE (SEQ ID NO: 84), heavy chain CDR2 with sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 with sequence: TKIYGNSFDY (SEQ ID NO: 40), CDR1 light chains with the sequence: KASQDVGTAV (SEQ ID NO: 85), CDR2 light chain with sequence: WASSRHN (SEQ ID NO: 46) and CDR3 light chain with sequence: QQYSRYPLT (SEQ ID NO: 48).

In some embodiments, the above-defined polynucleotide sequences encode a molecule selected from the group consisting of an antibody, an antibody fragment containing at least an antigen binding site, and an antibody conjugate containing said antibody or antibody fragment. Each embodiment is a separate embodiment of the present invention.

In some embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 68, or a variant thereof, having at least 90% sequence identity.

In some embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 72, or a variant thereof, having at least 90% sequence identity.

In some embodiments, a polynucleotide sequence encoding a heavy chain variable region of a monoclonal antibody comprises the sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 76, or a variant thereof, having at least 90% sequence identity.

In some embodiments, the polynucleotide sequence encoding the variable region of the light chain of a monoclonal antibody comprises the sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 70, or a variant thereof, having at least 90% sequence identity.

In some embodiments, the polynucleotide sequence encoding the variable region of the light chain of a monoclonal antibody comprises the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 74, or a variant thereof, having at least 90% sequence identity.

In some embodiments, the polynucleotide sequence encoding the variable region of the light chain of a monoclonal antibody comprises the sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 78, or a variant thereof, having at least 90% sequence identity.

In accordance with some embodiments, the present invention relates to a polypeptide comprising at least one sequence encoded by at least one polynucleotide sequence disclosed above.

In a further aspect, the present invention relates to a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof in accordance with the present invention. In some embodiments, the nucleic acid construct is a plasmid.

In some embodiments, the plasmid comprises the polynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 17, or SEQ ID NO: 33.

In some embodiments, the plasmid comprises the polynucleotide sequence set forth in SEQ ID NO: 68, SEQ ID NO: 72, or SEQ ID NO: 76.

In other embodiments, the plasmid comprises the polynucleotide sequence set forth in SEQ ID NO: 9, SEQ ID NO: 25, or SEQ ID NO: 41.

In other embodiments, the plasmid comprises the polynucleotide sequence set forth in SEQ ID NO: 70, SEQ ID NO: 74, or SEQ ID NO: 78.

In another aspect, the present invention provides a hybridoma cell capable of producing an antibody or antibody fragment containing specific CDR sequences and / or specific heavy and light chain variable regions as defined above.

In accordance with some embodiments, a hybridoma cell is provided comprising at least one polynucleotide sequence disclosed above.

In some embodiments, the hybridoma is capable of producing a monoclonal antibody comprising six complementarity determining regions (CDRs) sequences: (i) heavy chain CDR1 with the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), Heavy chain CDR2 with sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3 with sequence: PDGNYNALDY (SEQ ID NO: 82), light chain CDR1 with sequence: KASQDVGTAVT (SEQ ID NO: 12), light chain CDR2 with sequence : WASTRHT (SEQ ID NO: 14) and CDR3 light chain with sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chain CDR1 with sequence EYTMH (SEQ ID NO: 83), heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), light chain CDR1 with sequence: KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30) and light chain CDR3 with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) heavy chain CDR1 with sequence SNYWIE (SEQ ID NO: 84), heavy chain CDR2 with sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 with sequence: TKIYGNSFDY (SEQ ID NO: 40), CDR1 light chains with the sequence: KASQDVGTAV (SEQ ID NO: 85), CDR2 light chain with sequence: WASSRHN (SEQ ID NO: 46) and CDR3 light chain with sequence: QQYSRYPLT (SEQ ID NO: 48).

Antibodies or fragments thereof in accordance with the present invention can be attached to a cytotoxic component, a radioactive component or an identifiable component.

In accordance with another aspect, the present invention relates to a pharmaceutical composition containing, as an active ingredient, at least one antibody, antibody fragment or conjugates based on them that recognize PVR with high affinity and specificity, and optionally at least one pharmaceutically acceptable excipient , diluent, salt, or carrier.

In accordance with some embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof that is capable of binding to an epitope within a human PVR protein to which a monoclonal antibody selected from the group consisting of: (i) an antibody, herein identified as 4E5 (also referred to as PVR.07), with the variable region of the heavy chain SEQ ID NO: 2 and the variable region of the light chain SEQ ID NO: 10; (ii) an antibody, herein identified as 7D4 (also referred to as PVR.01), with a heavy chain variable region of SEQ ID NO: 18 and a light chain variable region of SEQ ID NO: 26; and (ii) an antibody, herein identified as 5B9 (also referred to as PVR.09), with a heavy chain variable region of SEQ ID NO: 34 and a light chain variable region of SEQ ID NO: 42.

In some embodiments, the pharmaceutical composition comprises a monoclonal antibody or antibody fragment thereof comprising six CDRs: (i) heavy chain CDR1 with the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80, heavy chain CDR2 with sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3 with sequence: PDGNYNALDY (SEQ ID NO: 82), light chain CDR1 with sequence: KASQDVGTAVT (SEQ ID NO: 12), light chain CDR2 with sequence: WASTRHT (SEQ ID NO: 14) and light chain CDR3 with sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chain CDR1 with sequence EYTMH (SEQ ID NO: 83), heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), light chain CDR1 with sequence: KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30) and light CDR3 chains with the sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) heavy chain CDR1 with sequence SNYWIE (SEQ ID NO: 84), heavy chain CDR2 with sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 with sequence: TKIYGNSFDY (SEQ ID NO: 40), CDR1 light chains with the sequence: KASQDVGTAV (SEQ ID NO: 85), CDR2 light chain with sequence: WASSRHN (SEQ ID NO: 46) and CDR3 light chain with sequence: QQYSRYPLT (SEQ ID NO: 48).

In accordance with some embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a heavy chain variable region with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 69, SEQ ID NO: 18, SEQ ID NO : 73, SEQ ID NO: 34 and SEQ ID NO: 77. Each embodiment is a separate embodiment of the present invention.

In accordance with some embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a light chain variable region with a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 71, SEQ ID NO: 26, SEQ ID NO : 75, SEQ ID NO: 42 and SEQ ID NO: 79. Each variant is a separate embodiment of the present invention.

In accordance with a specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a heavy chain variable region of the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 69 and a light chain variable region of the sequence set forth in SEQ ID NO : 10 or SEQ ID NO: 71.

In accordance with a specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a heavy chain variable region of the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 73 and a light chain variable region of the sequence set forth in SEQ ID NO : 26 or SEQ ID NO: 75.

In accordance with a specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a heavy chain variable region of the sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 77 and a light chain variable region of the sequence set forth in SEQ ID NO : 42 or SEQ ID NO: 79.

In accordance with some embodiments, the pharmaceutical composition comprises a combination of at least two antibodies or antibody fragments that recognize human PVR.

In accordance with still other embodiments, the pharmaceutical composition comprises one mAb or fragment that specifically binds PVR in accordance with the present invention and one mAb or fragment that specifically binds another antigen, such as a cell receptor, tumor antigen, or immunomodulatory protein.

Also provided are pharmaceutical compositions comprising at least one antibody, antibody fragment, or antibody-based conjugate according to the present invention for use in resuming NK cytotoxicity by inhibiting PVR binding to TIGIT expressed on the surface of NK cells.

In some embodiments, the antibody, antibody fragment, or antibody conjugate is capable of inhibiting the binding of human PVR to TIGIT expressed on the surface of T cells.

In accordance with some embodiments, the pharmaceutical composition of the present invention is for use in cancer immunotherapy or enhancing an immune response.

Cancer can be any cancer that expresses PVR. In some embodiments, the cancer overexpresses PVR.

In accordance with some embodiments of the present invention, the cancer is metastatic cancer. In accordance with some embodiments, the pharmaceutical composition of the present invention is for use in inhibiting the formation or spread of metastases, or reducing the total number of metastases in a subject.

In accordance with some embodiments of the present invention, the cancer is selected from the group consisting of melanoma, breast cancer, ovarian cancer, pancreatic cancer, colon and rectal cancer, colon cancer, cervical cancer, kidney cancer, lung cancer, thyroid cancer. gland, prostate cancer, brain cancer, kidney cancer, throat cancer, laryngeal carcinoma, bladder cancer, liver cancer, fibrosarcoma, endometrial cancer, glioblastoma, sarcoma, myeloid neoplasm, leukemia and lymphoma. Each embodiment is a separate embodiment of the present invention.

In some embodiments, the cancer is solid cancer. In accordance with some specific embodiments, the solid cancer is selected from the group consisting of melanoma (skin), lung, colon, breast, uterus, and kidney cancer. In accordance with specific embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, and liposarcoma.

In accordance with other embodiments, the cancer is hematologic cancer. In some embodiments, the hematologic cancer is myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, multiple myeloma, or myelodysplastic syndrome. Each embodiment is a separate embodiment of the present invention. In certain embodiments, the cancer is leukemia. In specific embodiments, the cancer is acute myeloid leukemia (AML).

In accordance with some embodiments, the pharmaceutical composition of the present invention is for use in the treatment of a viral infection.

In some embodiments, the viral infection is caused by a virus that binds to a target cell via PVR on the surface of the infected cell.

In accordance with some embodiments, the pharmaceutical composition of the present invention is for use in the treatment of an angiogenesis-associated disease or disorder. In accordance with certain embodiments, the angiogenesis-associated disease or disorder is selected from the group consisting of: cancer, ocular diseases (ocular diseases) associated with cell proliferation, retinal lesions, and inflammatory disease. Each embodiment is a separate embodiment of the present invention.

In another aspect, the present invention provides a method for inhibiting the binding of human PVR to at least one ligand by using a monoclonal antibody or antibody fragment as defined above.

In a further aspect, the present invention provides a method of enhancing an immune response in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a monoclonal antibody, antibody fragment or antibody conjugate as defined above.

In accordance with another aspect, the present invention relates to a method of treating cancer, comprising administering to a subject in need of a therapeutically effective amount of a pharmaceutical composition containing at least one antibody, antibody fragment or conjugate based on them that recognizes human PVR with high affinity and specificity , and are capable of inhibiting its binding to its ligand.

In some embodiments, the antibody in the pharmaceutical composition to be administered is selected from the group consisting of: (i) a monoclonal antibody comprising CDR sequences contained within the heavy chain variable region set forth in SEQ ID NO: 2 and CDR sequences comprised within the variable region of the light chain set forth in SEQ ID NO: 10; (ii) a monoclonal antibody containing the CDR sequences contained within the variable region of the heavy chain set forth in SEQ ID NO: 18 and the CDR sequences contained within the variable region of the light chain set forth in SEQ ID NO: 26; or (iii) a monoclonal antibody comprising the CDR sequences contained within the variable region of the heavy chain set forth in SEQ ID NO: 34 and the CDR sequences contained within the variable region of the light chain set forth in SEQ ID NO: 42.

In accordance with some specific embodiments, the monoclonal antibody in the pharmaceutical composition to be administered comprises: a heavy chain CDR1 comprising a sequence selected from the group consisting of GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); CDR2 of the heavy chain containing the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6); CDR3 of the heavy chain containing the sequence: PDGNYNALDY (SEQ ID NO: 82); CDR1 light chain containing the sequence: KASQDVGTAVT (SEQ ID NO: 12); CDR2 light chain containing the sequence: WASTRHT (SEQ ID NO: 14); and a light chain CDR3 containing the sequence: QQYSRYPYT (SEQ ID NO: 16). Each embodiment is a separate embodiment of the present invention.

In accordance with other specific embodiments, the monoclonal antibody in the pharmaceutical composition to be administered comprises: a heavy chain CDR1 comprising the sequence EYTMH (SEQ ID NO: 83); CDR2 of the heavy chain containing the sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); CDR3 of the heavy chain containing the sequence: VIPLEY (SEQ ID NO: 24); CDR1 light chain containing the sequence: KASQNVYTNVA (SEQ ID NO: 28); CDR2 light chain containing the sequence: SASYRYR (SEQ ID NO: 30); and a light chain CDR3 containing the sequence: QQYNSYPLA (SEQ ID NO: 32).

In accordance with other specific embodiments, the monoclonal antibody in the pharmaceutical composition to be administered comprises: a heavy chain CDR1 comprising the sequence SNYWIE (SEQ ID NO: 84); CDR2 of the heavy chain containing the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38); CDR3 of the heavy chain containing the sequence: TKIYGNSFDY (SEQ ID NO: 40); CDR1 light chain containing the sequence: KASQDVGTAV (SEQ ID NO: 85); CDR2 light chain containing the sequence: WASSRHN (SEQ ID NO: 46); and a light chain CDR3 containing the sequence: QQYSRYPLT (SEQ ID NO: 48).

In accordance with some embodiments of the present invention, a therapeutically effective amount results in a decrease in tumor size or metastases in a subject.

In accordance with some embodiments, a method for treating cancer comprises administering or administering at least one additional anti-cancer therapy. In accordance with certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, or immunotherapy.

In accordance with some embodiments, a method for treating cancer comprises administering a monoclonal antibody that recognizes human PVR with high affinity and specificity and an additional anti-cancer agent. In accordance with some embodiments, the additional anti-cancer agent is selected from the group consisting of: an immunomodulator, an activated lymphocyte, a kinase inhibitor, and a chemotherapeutic agent.

In other embodiments, the additional immunomodulator is an antibody, antibody fragment, or antibody conjugate that binds to an antigen other than human PVR.

In some embodiments, the additional immunomodulator is an antibody to an immune checkpoint molecule. In some embodiments, the additional immunomodulator is an antibody to an immune checkpoint molecule selected from the group consisting of human programmed cell death (PD-1) protein 1, PD-L1 and PD-L2, cell adhesion molecule 1, related embryonic tumor antigen (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, OX40 (also called CD134), immunoglobulin-like killer cell receptors (KIR), TIGIT, and any combination thereof. Each embodiment is a separate embodiment of the present invention.

In some embodiments, the anti-cancer agent is selected from the group consisting of: Erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine, carmustine, lomustine, chlorambucylate , chlormethine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, plikamycin, etoposide, teniposide, and any combination thereof. Each embodiment is a separate embodiment of the present invention.

In some embodiments, the anti-cancer agent is an epidermal growth factor receptor (EGFR) inhibitor. In some embodiments, the EGFR inhibitor is selected from the group consisting of: cetuximab (Erbitux®), panitumumab (Vectibix®), and necytumumab (Portrazza®). In some embodiments, the EGFR inhibitor is cetuximab (Erbitux®).

In accordance with some embodiments of the present invention, the subject is a human subject.

In accordance with some embodiments of the present invention, the use further comprises the use of an agent that down-regulates the activity or expression of an immune co-inhibiting receptor.

In some embodiments, the immune cell is a T cell.

In accordance with some embodiments of the present invention, the immune co-inhibiting receptor is selected from the group consisting of PD-1, TIGIT, DNAM-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55, LAIR1, SIGLEC10 and 2B4 ... Each embodiment is a separate embodiment of the present invention.

In accordance with one aspect, the present invention relates to a method for modulating the function and / or activity of the immune system, comprising modulating the binding of PVR to TIGIT using an antibody of the present invention.

In accordance with one aspect, the present invention provides a method for preventing or treating a viral infection caused by a virus that uses CD155 as a receptor to enter a cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a monoclonal antibody described herein to PVR. In some embodiments, the virus is selected from the group consisting of poliovirus, Coxsackie virus, adenovirus, and human immunodeficiency virus (HIV).

In another aspect, the present invention provides a method for treating an angiogenesis-associated disease or disorder. In accordance with certain embodiments, the angiogenesis-associated disease or disorder is selected from the group consisting of: cancer, ocular diseases (ocular diseases) associated with cell proliferation, retinal lesions, and inflammatory disease. Each embodiment is a separate embodiment of the present invention.

In accordance with some embodiments, a method for treating cancer comprises preventing or reducing the formation, growth, or spread of metastases in a subject by inhibiting angiogenesis.

In one aspect, the present invention provides a method for diagnosing or predicting a cancer or infectious disease in a subject, the method comprising determining the level of PVR expression in a biological sample from said subject using at least one antibody as described herein.

In accordance with another aspect, the present invention further provides a method for determining or quantifying the expression of PVR, the method comprising contacting a biological sample with an antibody or antibody fragment and measuring the degree of complexation, wherein the antibody or antibody fragment comprises complementarity determining regions (CDRs) selected from the group consisting of: (i) heavy chain CDR1 with the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), heavy chain CDR2 with the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy CDR3 chains with sequence: PDGNYNALDY (SEQ ID NO: 82), CDR1 light chain with sequence: KASQDVGTAVT (SEQ ID NO: 12), CDR2 light chain with sequence: WASTRHT (SEQ ID NO: 14) and CDR3 light chain with sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chain CDR1 with sequence EYTMH (SEQ ID NO: 83), heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), light chain CDR1 with sequence: KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30) and light chain CDR3 with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) heavy chain CDR1 with sequence SNYWIE (SEQ ID NO: 84), heavy chain CDR2 with sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 with sequence: TKIYGNSFDY (SEQ ID NO: 40), CDR1 light chains with the sequence: KASQDVGTAV (SEQ ID NO: 85), CDR2 light chain with sequence: WASSRHN (SEQ ID NO: 46) and CDR3 light chain with sequence: QQYSRYPLT (SEQ ID NO: 48).

In some embodiments, the detection and quantification methods can be performed in-vitro or ex-vivo , or can be used to diagnose conditions associated with PVR expression. Antibodies in accordance with the present invention can also be used for screening methods. For example, an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) can be adapted to measure levels of secreted or cell-associated polypeptide using antibodies and methods known in the art.

In accordance with some embodiments, a method for detecting the presence or quantification of PVR comprises the steps of:

i. incubating the sample with an antibody specific for PVR or an antibody fragment thereof containing at least an antigen binding site;

ii. identifying associated PVR using a detectable sample.

In accordance with some embodiments, the method further comprises the steps of:

iii. comparing the amount (ii) with a calibration curve obtained from the results from a reference sample containing a known amount of PVR; and

iv. calculating the amount of PVR in the sample from the calibration curve.

In accordance with some specific embodiments, the sample is a biological fluid.

In some embodiments, the method is performed in-vitro or ex-vivo.

Also provided is a kit for measuring the expression of PVR in a biological sample, containing at least one antibody or antibody fragment containing complementarity determining regions (CDRs) selected from the group consisting of: (i) CDR1 heavy chain with the sequence: GFDFSRYW (SEQ ID NO : 4) or RYWMT (SEQ ID NO: 80), heavy chain CDR2 with sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3 with sequence: PDGNYNALDY (SEQ ID NO: 82), light chain CDR1 with sequence: KASQDVGTAVT (SEQ ID NO: 12), light chain CDR2 with sequence: WASTRHT (SEQ ID NO: 14) and light chain CDR3 with sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chain CDR1 with sequence EYTMH (SEQ ID NO: 83), heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), light chain CDR1 with sequence: KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30) and light chain CDR3 with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) heavy chain CDR1 with sequence SNYWIE (SEQ ID NO: 84), heavy chain CDR2 with sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 with sequence: TKIYGNSFDY (SEQ ID NO: 40), CDR1 light chains with the sequence: KASQDVGTAV (SEQ ID NO: 85), CDR2 light chain with sequence: WASSRHN (SEQ ID NO: 46) and CDR3 light chain with sequence: QQYSRYPLT (SEQ ID NO: 48).

In accordance with one aspect, the present invention relates to a kit for detecting cancer, wherein the diagnostic kit comprises an antibody, or a fragment thereof, as disclosed herein.

In accordance with some embodiments, the present invention provides a method for diagnosing, assessing the severity or staging of an immune-related disease or proliferative disease, comprising determining the expression or activity of PVR in a sample from a subject using an antibody of the present invention or a fragment or conjugate thereof based on it, and comparing the expression or activity of PVR with a standard value of expression or activity of PVR. The specified standard value can be obtained from the analysis of a sample taken from a healthy subject, from the same subject at a different stage of the disease, or it can be determined from clinical studies of a large population of subjects.

Additional embodiments and the full range of applicability of the present invention will be apparent from the following detailed description. However, it should be understood that the detailed description and specific examples indicating the preferred embodiments of the present invention are provided solely as illustrative, since various changes and modifications within the spirit and scope of the present invention will be obvious to specialists in this field from this detailed description.

Brief Description of Drawings

Figures 1A-1C are graphs showing the correlation of PVR mRNA expression levels (high or low as indicated) with survival probability. Correlation was measured for lung cancer (Fig. 1A), breast cancer (Fig. 1B) and liposarcoma (Fig. 1C). Datasets of mRNA expression were obtained from the GEO site, and analyzed using the bioprofiling.de site, in the following form: dataset for lung cancer, GEO ID: GSE31210, dataset for breast cancer, GEO ID: GSE25055, and dataset for liposarcoma, GEO ID: GSE30929.

FIG. 2 is a schematic representation of the expression of a receptor on the surface of immune and tumor cells. TIGIT refers to a co-inhibiting receptor on the surface of many immune cells (eg, T cells); DNAM-1 (also called CD226) refers to an activating receptor on the surface of many immune cells (eg, T cells); Fc receptor refers to a potent activating receptor expressed primarily on the surface of NK cells, but also on the surface of myeloid cells, including neutrophils and macrophages; PVR refers to the inhibitory ligand TIGIT (weaker binding to DNAM-1) expressed by many tumor cells; Nectin-2 refers to the activating ligand DNAM-1 (negligible recognition of TIGIT) expressed by many tumor cells. In the case of binding of antibodies to PVR in accordance with the present invention, a double effect was observed: 1) an increase in the degree of destruction of tumor cells by means of Fc receptors; and 2) enhancing the activation of immune cells by blocking interactions with TIGIT.

Figures 3A-3D are graphs showing the results of FACS analysis of four obtained anti-PVR antibodies. The antibodies were shown to be effective in blocking direct binding of TIGIT-Fc to the tumor cell line. FIG. 3A shows the results for non-blocking anti-PVR antibody (anti-PVR mAb 2G3, also referred to as hPVR.17). Figures 3B-3D show that three of the resulting antibodies, namely 5B9 (also called hPVR.09), 7D4 (also called hPVR.01) and 4E5 (also called hPVR.07) respectively, are blocking mAbs to PVR, as shown to block the binding of TIGIT-Ig. Hybridoma culture media (5 μl / well) were added to HepG2 cells. TIGIT-Fc was applied at 2 μg / well to give a final concentration of 20 μg / ml, and levels of cell-bound TIGIT were measured by FACS after adding fluorescently labeled Ab to the Fc. The filled-in portions of the histogram represent background staining with the Fc binding reagent.

FIG. 4 is a graph showing how blocking PVR-TIGIT interactions with anti-PVR mAb 7D4 (also called hPVR.01) increases NK killing of the MDA-MB-231 human cell line (breast adenocarcinoma). Specific killing was calculated from [35S] -methionine secretion from target cells. Control (Ctrl) is unrelated mouse IgG. P-value = 0.0056.

FIG. 5 is a graph showing that blocking PVR-TIGIT interactions with the anti-PVR mAb 4E5 (also called hPVR.07) increases NK killing of the HepG2 human cancer cell line (hepatocellular carcinoma). None - no HepG2 killing was observed without mAb; 4E5 to PVR - elimination of HepG2 with mouse 4E5mIgG1 to PVR (without activation of the human Fc receptor); 4e5hIgG1 to PVR - elimination of HepG2 with 4E5-hIgG1 to PVR (activation of human Fc receptor), Erbitux (PC) - mAb Erbitux as a positive control (antibody to EGFR). All mAbs were used at a concentration of 10 μg / ml. P-values: 4E5 to PVR - 0.04, 4e5hIgG1 to PVR - 0.000746 and Erbitux (positive control) - 0.003219.

Figures 6A-6O are graphs showing that human tumor cell lines express PVR and Nectin-2. Melanoma cells (Figures 6A-E), breast cancer cells (Figures 6F-H), colon and rectal cancer cells (Fig. 6I), kidney cancer cells (Fig. 6J), lung cancer cells (Fig. 6K), prostate cancer cells (Fig. 6L), brain tumor cells (Fig. 6M) and hepatocellular carcinoma cells (Figures 6N-O) all express PVR and Nectin-2. The mAb was used at a concentration of 0.2 μg / well: a commercial anti-Nectin-2 mAb and an anti-PVR mAb 4E5 (also called hPVR.07).

Figures 7A-7C are graphs of FACS analysis results showing that PVR is the main ligand of TIGIT. FIG. 7A illustrates that HepG2 cells (human hepatocellular carcinoma cells) express PVR and Nectin-2. FIG. 7B illustrates that purified anti-PVR mAb 4E5 (also called hPVR.07) (0.15 μg) almost completely (over 97%) blocks TIGIT-Ig binding (2 μg / ml), regardless of the fact that these cells also express Nectin-2. FIG. 7C illustrates that CHO cells express hNectin-2 at a high level. FIG. 7D shows no staining for the same CHO cells as in FIG. 7C, with all anti-PVR mAbs, which means that there is no direct recognition of Nectin-2 by the anti-PVR mAb, and thus the blocking of TIGIT binding observed in FIG. 7B cannot be explained by blocking Nectin-2, rather it is the result of direct blocking of PVR.

Figures 8A-8C show similar binding efficiencies for all anti-PVR mAbs to human PVR. All three blocking clones provided similar (less than 10% difference) binding to both endogenous PVR-expressing HepG2 cells (FIG. 8A) and hPVR-overexpressing B16-hPVR cells (FIG. 8B). Binding was also studied using the African green monkey Vero cell line (Fig. 8C), which expresses a PVR protein with 93% similarity to human PVR. In this case, different indices of staining intensity were observed for different Ab. In all cases, 0.2 μg of each mAb was used.

FIG. 9 is a graph showing that anti-PVR antibodies of the present invention do not recognize canine PVR. Human TIGIT-Fc (10 μg / ml) is highly cross-reactive and binds to the canine MDCK cell line. None of the anti-PVR mAbs were able to bind to these cells, indicating that they did not recognize canine PVR.

Figures 10A-10D show that Nectin-2 binds preferentially to DNAM-1 rather than TIGIT. Cells overexpressing either PVR or Nectin-2 were stained using the indicated antibody concentrations. FIG. 10A illustrates binding of TIGIT-Fc to Nectin-2 expressing cells; in fig. 10B illustrates the binding of DNAM-FC to Nectin-2 expressing cells. FIG. 10C illustrates the binding of TIGIT to PVR expressing cells. FIG. 10D illustrates the binding of DNAM-1 to PVR expressing cells.

FIG. 11 shows the effect of anti-PVR antibodies on T cell proliferation. Human PBMCs were labeled with CFSE and incubated with target cells in the presence of the indicated antibodies. The results are presented as the fold of the increased degree of proliferation compared to the control. Results are pooled from 7 experiments for a total of 10 healthy donors. P-values: for mAb 4E5 - 0.000241, for mAb 5B9 - 1.96E ^-05 , for anti-PD-1 antibody - 0.016303, for anti-CTLA4 antibody - 0.000171 and for 4E5hIgG1 - 0.008176.

FIG. 12 shows the combined effect of anti-PVR antibodies and other antibodies on T cell proliferation. Human PBMCs were labeled with CFSE and incubated with target cells in the presence of this antibody combination. The results are presented as the fold of the increased degree of proliferation compared to the control. Results include 7 independent experiments on 12 healthy donors. P-values: anti-PD1 + anti-CTLA antibody - 47.54E- ⁰³ , anti-PD-1 + 4E5 antibody - 7.02E ^-02 , anti-PD-1 + 5B9 antibody - 1.11E ^-04 , anti-CTLA4 + antibody 4E5 - 1.37E ^-03 , anti-CTLA4 + 5B9 - 5.47E ^-06 .

FIG. 13 shows the specific effect of anti-PVR antibodies on CD8 T cell proliferation. Human PBMCs were labeled with CFSE and incubated with target cells (A549) in the presence of the indicated antibodies. CD8 positive cells were counted by FACS after 9-12 days of culture. The results are presented as the fold of the increased degree of proliferation compared to the control. Results include 2 independent experiments on healthy PBMC donors.

FIG. 14 shows the ratio of CD8 cells to CD4 cells after induction with different antibodies. Human PBMCs were labeled with CFSE and incubated with target cells (A549) in the presence of the indicated antibodies. Cells were counted after 9-12 days of culture. Results are pooled from 2 experiments for a total of 2 healthy donors.

FIG. 15 shows the effect of anti-PVR antibodies on NK cell degranulation. Human NK cells were incubated with MDA-MB-231 cells (triple negative breast cancer cell line) in the presence of these antibodies. Results are presented for 7 independent experiments performed on 5 different healthy NK cell donors. P-values: PVR4E5 - 0.005063, PVR5B9 - 0.00374, PVR7D4 - 0.019448, PVR4E5-hIgG1 - 2.03E ^-05 , PVR5B9-hIgG1 - 1.45E ^-05 , PVR7D4-hIgG1 5.8E ^-05 ...

Figures 16A-16D show the effect of anti-PVR antibodies on tumor cell survival in the absence of immune cells. The survival of A549 cells (Fig.16A), U373 cells (Fig.16B), HCT116 cells (Fig.16C) and Mel-624 (Fig.16D) was studied using the MTT cell survival assay in the presence of 50 micrograms / ml of the indicated mAb on for 24 hours. The level of significance was calculated using the one-tailed Student's t test, * <0.05, ** <0.03, and *** <0.02.

Detailed Disclosure of the Present Invention

The present invention relates to monoclonal antibodies specific for the human poliovirus receptor (PVR). The present invention also relates to the preparation and use of mAbs as therapeutic agents. In particular, the mAbs of the present invention can be used, alone or in combination with other agents, to reactivate and enhance the tumor-killing activity of immune cells and as diagnostic reagents.

The term "antigen" as used herein refers to a molecule or portion of a molecule capable of eliciting the formation of an antibody and specifically binding to an antibody. An antigen can have one or more than one epitope. The above specific binding means that the antigen will interact, with high selectivity, with its corresponding antibody, but not with many other antibodies, the formation of which can be induced by other antigens. The antigen, in accordance with some embodiments of the present invention, is a PVR protein.

The term "PVR" in the context of this document refers to the poliovirus receptor, also known as CD155 (cluster of differentiation 155). PVR is a transmembrane glycoprotein with an N-terminal signal sequence, three extracellular immunoglobulin (Ig) -like domains, a transmembrane domain, and a cytoplasmic tail. It has a molecular weight of about 80 kDa and a structure consisting of three Ig-like domains, namely the outermost V-like domain, followed by two C2-like domains. An exemplary PVR in accordance with the present invention is presented in GenBank under accession numbers: NP_001129240.1, NP_001129241.1, NP_001129242.2 and NP_006496.4. These poliovirus receptors share a common extracellular domain sequence and therefore can be targeted by the affinity binding component of the present invention.

The term "antigenic determinant" or "epitope" as used herein refers to the region of an antigen molecule that specifically interacts with a particular antibody. Peptide sequences derived from an epitope can be used, alone or in combination with a component as a carrier, using methods known in the art, to immunize animals and to generate additional polyclonal or monoclonal antibodies. Isolated peptides derived from an epitope can be used in diagnostic methods to detect antibodies.

It should be noted that affinity can be quantified using known methods such as surface plasmon resonance (SPR) (described in Scarano S, Mascini M, Turner AP, Minunni M. Surface plasmon resonance imaging for affinity-based biosensors. Biosens Bioelectron. 2010, 25: 957-66), and can be calculated using, for example, the dissociation constant, Kd, such that a lower Kd represents a higher affinity.

Antibodies or a fragment thereof in accordance with the present invention bind to an epitope in the hPVR. In particular, antibodies bind to an epitope within amino acids 1-343 of the PVR set forth under NP_006496.4.

Antibodies, or immunoglobulins, contain two heavy chains linked together by disulfide bonds and two light chains, with each light chain connected to a corresponding heavy chain by disulfide bonds in a "Y" configuration. As a result of proteolytic cleavage of the antibody, the Fv (variable region fragment) and Fc (crystallizing fragment) domains are formed. Antigen binding domains, Fab, include regions in which the polypeptide sequence varies. The term F (ab ') ₂ means two Fab' arms connected together by disulfide bonds. The central axis of the antibody is called the Fc fragment. Each heavy chain contains at one end a variable domain (V _H ), followed by a number of constant domains ( _CH ). Each light chain contains a variable domain (V _L ) at one end and a constant domain (C _L ) at the other end, with the variable domain of the light chain parallel to the variable domain of the heavy chain, and the constant domain of the light chain parallel to the first constant domain of the heavy chain ( CH1). The variable domains of each pair of light and heavy chains form an antigen-binding site. The light and heavy chain domains share the same general structure, and each domain contains four framework regions, the sequences of which are relatively conserved, connected by three hypervariable domains known as complementarity determining regions (CDRs 1-3). These domains provide the specificity and affinity of the antigen binding site.

The identification or determination of CDRs from a given variable sequence of a heavy or light chain is usually performed using one of several methods known in the art. For example, this definition is made in accordance with Kabat (Wu TT and Kabat EA, J Exp Med , 1970; 132: 211-50) and IMGT (Lefranc MP, et al., Dev Comp Immunol , 2003, 27: 55-77) ...

When the term “sequence CDR” or a similar term is used, it includes variants where the CDR contains the specified sequence, as well as variants where the CDR consists of the specified sequence.

The antigenic specificity of an antibody is due to the hypervariable region (HVR), namely the unique CDR sequences of both light and heavy chains, which together form an antigen-binding site.

The heavy chain isotype (gamma, alpha, delta, epsilon, or mu) determines the class of immunoglobulin (IgG, IgA, IgD, IgE, or IgM, respectively). The light chain belongs to one of two isotypes (kappa, κ, or lambda, λ). Both isotypes are found among all classes of antibodies.

The term "antibody" is used in its broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, and antibody fragments of a length sufficient to exhibit the desired biological activity, namely, binding to human PVR.

An antibody or antibodies in accordance with the present invention include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as fragments thereof resulting from proteolysis, such as Fab or F (ab ') ₂ fragments. Single chain antibodies are also encompassed within the scope of the present invention.

Antibody fragments

"Antibody fragments" contain only a portion of the intact antibody, including usually the antigen-binding site of the intact antibody, and therefore retain the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) a Fab fragment with the VL, CL, VH and CH1 domains; (ii) a Fab 'fragment, which is a Fab fragment with one or more cysteine residues at the C-terminus of the CH1 domain; (iii) an Fd fragment with the VH and CH1 domains; (iv) an Fd'-fragment with the VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (v) an Fv fragment with the VL and VH domains of one arm of an antibody; (vi) a dAb fragment (Ward et al., Nature 1989, 341, 544-546), which consists of a VH domain; (vii) isolated CDR regions; (viii) F (ab ') ₂ fragments, a bivalent fragment, including two Fab' fragments connected by a disulfide bridge in the hinge region; (ix) single chain antibody molecules (e.g. single chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. (USA) 1988, 85, 5879-5883); (x) "diabodies" with two antigen binding sites containing a heavy chain variable domain (VH) fused to a light chain variable domain (VL) within the same polypeptide chain (see, for example, EP 404.097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) "linear antibodies" containing a pair of tandem Fd segments (VH-CH1-VH-CH1), which, together with the complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng., 1995, 8, 1057-1062; and U.S. Patent No. 5,641,870).

Various techniques have been developed to obtain antibody fragments. Traditionally, such fragments were obtained by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24 : 107-117 (1992) and Brennan et al., Science, 229 : 81 (1985)). However, at present, such fragments can be produced by recombinant host cells. For example, antibody fragments can be isolated from an antibody phage library. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab ') ₂ fragments (Carter et al., Bio / Technology 10 : 163-167 (1992)). According to another approach, F (ab ') ₂ fragments can be isolated directly from the culture of recombinant host cells. Other techniques for obtaining antibody fragments will be apparent to the practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv).

Single-chain antibodies can be single-chain composite polypeptides characterized by antigen-binding properties and containing amino acid sequences homologous to or similar to the variable regions of the light and heavy chains of immunoglobulins, i.e., linked V _H -V _L or single-chain Fv (scFv). The techniques for producing single chain antibodies (US Pat. No. 4,946,778) can be modified to produce single chain antibodies to PVR.

The term "monoclonal antibody" (mAb), as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that make up the population are identical except for possible naturally occurring mutations, which may be in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. In addition, unlike polyclonal antibody fractions, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring the antibody to be produced by any particular method. mAbs can be prepared by methods known to those skilled in the art. For example, monoclonal antibodies to be used in accordance with the present invention can be obtained using the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or can be obtained using methods based on recombinant DNA technology (see, for example , US patent No. 4816567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol. 1991 222: 581-597.

The design and development of recombinant monovalent antigen binding molecules derived from monoclonal antibodies through the rapid identification and cloning of functional heavy (VH) and variable light (VL) chain genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria are described in Fields et at. 2013, 8 (6): 1125-48.

The mAbs of the present invention can belong to any class of immunoglobulins, including IgG, IgM, IgE, IgA, and IgD. The mAb-producing hybridoma can be cultured in-vitro or in-vivo . High titers of mAbs can be obtained by in-vivo production in which cells of individual hybridomas are injected intraperitoneally into Balb / c mice that have undergone primary stimulation with pristine to produce ascites fluid containing high concentrations of the desired mAb. mAbs can be purified from such ascites fluid or from culture supernatants using methods well known to those skilled in the art.

Also included are anti-idiotypic antibodies that are specifically immunoreactive against the hypervariable regions of the antibody of the present invention.

The present invention relates to a monoclonal antibody or antibody fragment comprising an antigen binding domain (ABD) that contains three light chain CDRs and three heavy chain CDRs, said ABD having at least 90% sequence identity or sequence similarity to the ABD of a mouse monoclonal antibody comprising: (I) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO: 69, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 71 (herein identified as 4E5 or hPVR.07); (ii) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO: 73, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 75 (herein identified as 7D4 or hPVR.01); or (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 77 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79 (herein identified as 5B9 or hPVR.09). Such an antibody may have an ABD domain characterized by at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, at least 99% identity or similarity sequence or 100% sequence identity with the corresponding ABD 4E5, 7D4 or 5B9.

Sequence identity is the number of amino acids or nucleotides that match exactly for two different sequences. The sequence similarity allows for conservative substitution of amino acids, defined as identical amino acids.

The present invention also relates to variants of the antibody molecules in accordance with the present invention with conservative amino acid substitutions. You can also obtain variants in accordance with the present invention, which retain the overall molecular structure of the encoded proteins. Given the properties of the individual amino acids forming the disclosed protein products, some suitable substitutions will be known to the person skilled in the art. Amino acid substitutions, ie, "conservative substitutions", can be made, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues in question. The term "antibody analog" in the context of this document refers to an antibody obtained from another antibody by making one or more conservative amino acid substitutions.

The term "antibody variant" as used herein refers to any molecule comprising an antibody of the present invention. For example, fusion proteins in which an antibody or antigen-binding fragment thereof is linked to another chemical compound are considered a variant antibody.

Analogs and variants of antibody sequences are also included in the scope of this application. These include, without limitation, conservative and non-conservative substitution, insertion and deletion of amino acids within a sequence. Such modification and the resulting antibody analog or variant are within the scope of the present invention provided that they provide or even improve the binding of the antibody to human PVR.

Conservative amino acid substitutions known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions involve the substitution of one amino acid for another with a functional group or side chain of the same type, for example, aliphatic, aromatic, positively charged, negatively charged. Such substitutions can provide increased oral bioavailability, penetration and targeting of specific cell populations, immunogenicity, etc. One skilled in the art will understand that individual substitutions, deletions or additions in a peptide, polypeptide or protein sequence that provide a change, the addition or removal of one amino acid or a small percentage of amino acids in the encoded sequence refers to a "conservatively modified variant" where the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables showing functionally similar amino acids are known in the art. For example, according to one table known in the art, each of the following six groups contains amino acids that are conservative substitutions for each other:

1) Alanine (A), serine (S), threonine (T);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), glutamine (Q);

4) Arginine (R), lysine (K);

5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

It should be noted that variant chain sequences are determined using sequencing methods using specific primers. Different sequencing methods used in relation to the same sequence can give slightly different sequences, due to technical aspects and the use of different primers, especially at the ends of the sequences. Therefore, in the present application, different variants of the sequences of the variable regions of the chains of anti-PVR antibodies are indicated.

The terms "molecule with an antigen-binding antibody moiety" and "antigen-binding fragments" in the context of this document are meant to include not only intact immunoglobulin molecules of any isotype and obtained using any animal cell line or microorganism, but also their antigen-binding reactive moiety, including without limitations Fab fragment, Fab 'fragment, F (ab') ₂ fragment, variable portion of their heavy and / or light chains, Fab mini-antibodies (see, for example, WO 93/15210, US Patent Application Publication 08 / 256,790, WO 96/13583, US Patent Application Publication 08/817788, WO 96/37621, US Patent Application Publication 08/999554) and single chain antibodies comprising such a reactive moiety, as well as any other type of molecule in which such a reactive portion of the antibody was physically inserted. Such molecules can be prepared using any known technique, including, but not limited to, enzymatic digestion, peptide synthesis, or recombinant techniques.

Monoclonal antibodies herein include, in particular, "chimeric" antibodies in which part of the heavy and / or light chain is identical or homologous to the corresponding antibody sequences derived from a particular species, or belonging to a particular class or subclass of antibodies, while the rest of the chain ( -e) is identical or homologous to the corresponding antibody sequences, derived from another species, or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (US patent No. 4816567; and Morrison et al ., Proc Natl Acad Sci USA 81 : 6851-6855 (1984)). In addition, complementarity determining region (CDR) grafting can be performed to alter certain properties of the antibody molecule, including affinity or specificity. A non-limiting example of CDR grafting is disclosed in US Pat. No. 5,225,539.

Chimeric antibodies are molecules, different portions of which are derived from different animal species, such as those containing a murine mAb derived variable region and a human immunoglobulin constant region. Antibodies that contain variable region framework residues primarily from a human antibody (called an acceptor antibody) and CDRs mainly from a mouse antibody (called a donor antibody) are also called humanized antibodies. Chimeric antibodies are used primarily to reduce immunogenicity in use and to increase yield in production, for example, in the case where mouse mAbs are produced by hybridomas with a higher yield, but are characterized by higher immunogenicity in humans, whereby chimeric human / mouse mAbs are used. Chimeric antibodies and methods for their preparation are known in the art (e.g., PCT patent applications WO 86/01533, WO 97/02671, WO 90/07861, WO 92/22653 and US patents 5693762, 5693761, 5585089, 5530101 and 5225539) ...

In some specific embodiments, the monoclonal antibody is a chimeric monoclonal antibody.

In some embodiments, the chimeric antibody comprises constant regions of human origin.

In some embodiments, the human constant regions of the chimeric antibody are selected from the group consisting of: human IgG1, human IgG2, human IgG3, and human IgG4.

In accordance with a specific embodiment, the chimeric monoclonal antibody or fragment thereof comprises a constant region of the human IgG1 subclass.

In accordance with a particular embodiment, a chimeric monoclonal antibody is provided that recognizes a PVR comprising a set of six CDRs selected from the group consisting of: (i) SEQ ID NO: 4 or 80, 6 or 81, 8 or 82, 12, 14 and 16; (ii) SEQ ID No: 20 or 83, 22, 24, 28, 30 and 32; and (iii) SEQ ID No: 36 or 84, 38, 40, 44 or 85, 46 and 48.

Pharmacology

In pharmaceutical and dosage formulations, the active agent is preferably used in conjunction with one or more pharmaceutically acceptable carriers and optionally any other therapeutic ingredients. The carrier (s) must be pharmaceutically acceptable in the sense that it is (are) compatible with the other ingredients of the formulation and is (are) not unduly harmful to the respective recipient. The active agent is provided in an amount effective to achieve the desired pharmacological effect as described above and in a dosage suitable to achieve the required exposure.

Typically, antibodies and fragments and conjugates based on the present invention, containing an antigen-binding portion of the antibody or containing another polypeptide, including a peptidomimetic, will be suspended in sterile saline solution for therapeutic applications. Pharmaceutical compositions can alternatively be formulated with the possibility of controlled release of the active ingredient (a molecule containing the antigen-binding portion of the antibody) or to prolong its stay in the patient's body. Many suitable drug delivery systems are known and include, for example, implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, etc. Controlled release formulations can be prepared by using polymers to adsorb the molecule in accordance with the present invention or linking it into a complex. For example, biocompatible polymers include matrices of an ethylene-vinyl acetate copolymer and matrices of a polyanhydride copolymer of stearic acid-sebacic acid dimer. The rate of release of a molecule in accordance with the present invention, i.e., an antibody or an antibody fragment, from such a matrix depends on the molecular weight of the molecule, the amount of the molecule in the matrix and the size of the dispersed particles.

The pharmaceutical composition of the present invention can be administered by any suitable route, for example, orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intraarterially, intraarticularly, intralesionally, intratumorally, or parenterally. Typically, intravenous (i.v.) administration is used to deliver antibodies.

Those of skill in the art will understand that a therapeutically effective amount of a molecule in accordance with the present invention will depend, inter alia, on the dosing regimen, the single dose of the molecule administered, whether the molecule is administered in combination with other therapeutic agents, immune status and health conditions. the patient, the therapeutic activity of the administered molecule, its stability in the circulatory system and the opinion of the attending physician.

As used herein, the term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduction in tumor size; inhibition (i.e., slowing to some extent, and preferably stopping) the infiltration of cancer cells into peripheral organs; inhibition (ie, slowing to some extent, and preferably stopping) tumor metastasis; inhibiting, to some extent, tumor growth; and / or alleviating, to some extent, one or more of the symptoms associated with the disorder. If a drug can prevent the growth and / or destruction of existing cancer cells, it can be considered cytostatic and / or cytotoxic. In the case of cancer therapy, efficacy in vivo can be measured, for example, by assessing survival time, time to disease progression (TTP), response rates (RR), response duration and / or quality of life.

Cancers amenable to treatment using the present invention include, but are not limited to: carcinoma, lymphoma, blastoma, sarcoma, and leukemia, or forms of lymphoedema. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell lung carcinoma), peritoneal cancer, hepatocellular cancer, stomach cancer, or gastric cancer (including malignant gastrointestinal tumor), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colon and rectal cancer, endometrial or uterine carcinoma, carcinoma salivary glands, cancer or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, and various types of head and neck cancer, and B-cell lymphoma (including non-Hodgkin's lymphoma (NHL) low grade / follicular non-Hodgkin's lymphoma y; small cell lymphocytic (SL) NHL; NHL intermediate grade / follicular NHL; diffuse intermediate grade NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; NHL with high grade uncleaved cells; NHL with massive lymph node involvement; lymphoma from cells of the marginal zone; AIDS-associated lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disease (PTLD), as well as abnormal vascular cell proliferation associated with phakomatoses, edema (eg, that associated with brain tumors) and Meigs syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colon and rectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft sarcoma tissues, Kaposi's sarcoma, carcinoid tumor, head and neck cancer, melanoma, ovarian cancer, mesothelioma and multiple myeloma. Cancer conditions amenable to the treatment of the present invention include metastatic cancers.

In accordance with other embodiments, the pharmaceutical composition of the present invention is for use in the treatment of cancer characterized by overexpression of PVR. The types of malignant tumors associated with overexpression of PVR can be identified using known databases such as The Cancer Genome Atlas (TCGA). In accordance with certain embodiments, the cancer is selected from the group consisting of adrenocortical carcinoma (ACC), chromophobic renal cell carcinoma (KICH), hepatocellular carcinoma of the liver (LIHC), adenocarcinoma of the colon and rectum (COAD, READ), adenocarcinoma of the pancreatic duct (PAAD), pheochromocytomas and paragangliomas (PCPG), papillary renal carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), endometrial carcinoma of the uterine body (UCEC) uterine cancer (CESC), skin melanoma (SKCM), mesothelioma (MESO), urothelial bladder cancer (BLCA), clear cell kidney carcinoma (KIRC), lung squamous cell carcinoma (LUSC), uterine carcinosarcoma (UCS), sarcoma ( ovarian cystadenocarcinoma (OV), papillary thyroid carcinoma (THCA), glioblastoma multiforme (GBM), breast cancer (BRCA), low-grade glioma (LGG) and diff nodular large B cell lymphoma (DLBC). Each embodiment is a separate embodiment of the present invention.

The molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, etc., and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if necessary, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, agents that maintain the pH in a certain range.

The pharmaceutical composition according to the present invention can be administered together with an antineoplastic composition.

The term "treatment" in the context of this document refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those with a pre-existing disorder as well as those in whom the disorder is preventable.

The terms "cancer" and "malignant" refer to or describe the physiological condition in mammals, which is usually characterized by uncontrolled cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include melanoma, lung cancer, thyroid cancer, breast, colon, prostate, liver, bladder, kidney, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian cancer, malignant tumor of the uterus, sarcoma, malignant tumor of the biliary system or malignant tumor of the endometrium.

In accordance with some embodiments, a method for treating cancer comprises administering a pharmaceutical composition as part of a treatment regimen comprising administering at least one additional anti-cancer agent.

In some embodiments, the anticancer agent is selected from the group consisting of an antimetabolite, a mitosis inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, an asparaginase, an alkylating agent, an antitumor antibiotic, and combinations thereof. Each embodiment is a separate embodiment of the present invention.

In some embodiments, the antimetabolite is selected from the group consisting of cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. In some embodiments, the mitosis inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine. In some embodiments, the topoisomerase inhibitor is selected from the group consisting of topotecan and irenetecan. In some embodiments, the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, chlormethine, melphalan, thiotepa, dacarbazine, and procarbazine. In some embodiments, the anti-tumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin. In some embodiments, the topoisomerase II inhibitor is selected from the group consisting of etoposide and teniposide. Each embodiment is a separate embodiment of the present invention.

In some specific embodiments, the additional anti-cancer agent is selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof. Each embodiment is a separate embodiment of the present invention.

Monoclonal antibodies in accordance with the present invention can be used as part of combination therapy with at least one anti-cancer agent. In some embodiments, the additional anti-cancer agent is an immunomodulator, an activated lymphocyte, a kinase inhibitor, or a chemotherapeutic agent.

In some embodiments, the anti-cancer agent is an immunomodulator, whether agonist or antagonist, such as an antibody to an immune checkpoint molecule.

Checkpoint blocking immunotherapy has been shown to be a promising measure in cancer treatment. Signaling pathways involving checkpoints of the immune response consist of a number of costimulatory and inhibitory molecules that function in concert to maintain self-tolerance and protect tissues from damage by the immune system under physiological conditions. Tumors take advantage of certain signaling pathways involving checkpoints to evade the immune system. Therefore, inhibition of such signaling pathways has proven to be a promising anticancer treatment strategy.

Antibody to cytotoxic T-lymphocyte associated protein 4 (CTLA-4) ipilimumab (approved in 2011) was the first immunotherapeutic agent for which treatment benefit has been shown in patients with malignant tumors. The specified antibody interferes with inhibitory signals during the presentation of antigen to T cells. The anti-programmed cell death protein 1 (PD-1) antibody pembrolizumab (approved 2014) blocks signaling by the T-cell-expressed PD-1 receptor, which downregulates the immune response. An additional agent that binds to PD-1 was registered with regulatory authorities in 2014 for the treatment of non-small cell lung cancer (NSCLC). As part of active research to date, many other immune response checkpoints are being studied, including: CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, Lymphocyte Activation Gene 3 (LAG3), CD137, OX40 (also called CD134) and immunoglobulin-like killer cell receptors (KIR).

In accordance with some specific embodiments, the immunomodulator is selected from the group consisting of: an antibody that inhibits CTLA-4, an antibody to human programmed cell death (PD-1) protein 1, an anti-PD-L1 and PD-L2 antibody, an activated cytotoxic lymphocyte, lymphocyte activating agents, antibodies to CEACAM, antibodies to TIGIT, and an inhibitor of the RAF / MEK signaling pathway. Each embodiment is a separate embodiment of the present invention. In some specific embodiments, the additional immunomodulator is selected from anti-PD-1 mAb, anti-PD-L1 mAb, anti-PD-L2 mAb, anti-CEACAM1 mAb, anti-CTLA-4 mAb, anti-TIGIT mAB, interleukin 2 (IL-2) or lymphokine-activated killer (LAK) cells.

In accordance with other embodiments, the additional anti-cancer agent is a chemotherapeutic agent. A chemotherapeutic agent, which may be administered together with an antibody of the present invention, or alone, may include any such agent known in the art to exhibit anti-cancer activity, including but not limited to: mitoxantrone, topoisomerase inhibitors, vinca spindle venom: vinblastine, vincristine , vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: chlormethine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabine; podophyllotoxins: etoposide, irinotecan, topotecan, dacarbazine; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen, leuprorelin, flutamide and megestrol acetate.

In some embodiments, the chemotherapeutic agent is selected from alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitor, interferons, coordination complexes, substituted anthracenedione, methylhydrazine derivatives, a drug that suppresses the synthesis of adrenal cortex hormones, adrenocorticosteroids, progestins, estrogens, antiestrogen, androgens, antiandrogen and gonadoliberin analog. In accordance with another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irenetecan, oxaliplatin, capecitabine, paclitaxel, and docetaxel. One or more chemotherapeutic agents can be used.

In some embodiments, the pharmaceutical composition of the present invention is for use in the treatment of cancer or for use in enhancing an immune response.

The term "enhancing the immune response" refers to increasing the reactivity of the immune system and inducing or prolonging its memory. The pharmaceutical composition according to the present invention can be used to stimulate the immune system as a result of vaccination. Thus, in one embodiment, the pharmaceutical composition can be used to enhance vaccination efficacy.

In certain embodiments, the cancer is selected from lung cancer, thyroid cancer, breast cancer, colon cancer, melanoma, prostate cancer, liver, bladder, kidney, cervix, pancreas, leukemia, lymphoma, myeloid neoplasm, ovarian cancer , cancer of the uterus, sarcoma, cancer of the biliary system and cancer from endometrial cells. Each embodiment is a separate embodiment of the present invention.

In accordance with some embodiments, a pharmaceutical composition comprising at least one antibody or fragment thereof in accordance with the present invention and a pharmaceutical composition comprising an additional immunomodulator or kinase inhibitor is used in the treatment of cancer by separate administration.

In accordance with another aspect, the present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a monoclonal antibody or antibody fragment in accordance with the present invention.

The term "effective amount" as used herein refers to a sufficient amount of a monoclonal antibody or antibody fragment that, when administered to a subject, will produce the intended therapeutic effect. The effective amount required to achieve a therapeutic endpoint may depend on a number of factors, including, for example, the particular type of tumor and the severity of the patient's condition, and whether the combination is additionally used in conjunction with radiation. An effective amount (dose) of active agents in the context of the present invention should be sufficient to provide a favorable therapeutic response in a subject over an extended period, including, but not limited to, inhibition of tumor growth, a decrease in the rate of tumor growth, prevention of tumor growth and metastasis, and increased survival.

The toxicity and therapeutic efficacy of the compositions described herein can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the IC50 (concentration that provides 50% inhibition) and the maximum tolerated dose for the compound of interest. The data obtained from such analyzes using cell culture and animal studies can be used in formulating dosages for use in humans. The dosage may vary depending on, inter alia, the dosage form used, the dosage regimen chosen, the composition of the agents used for treatment, and the route of administration used, among other relevant factors. The exact formulation, route of administration and dosage can be selected by the attending physician taking into account the condition of the patient. Depending on the severity and responsiveness of the condition to be treated, dosing may also be a single administration of a slow release formulation, with the course of treatment lasting from several days to several weeks, or until a cure or relief of the disease is achieved. The amount of composition administered will depend, as already mentioned, on the subject to be treated, the severity of the disease, the route of administration, the judgment of the attending physician, and all other relevant factors.

The term "use" or "administration" of a substance, compound, or agent to a subject is intended to be performed using one of a number of methods known to those of skill in the art. For example, the compound or agent can be administered enterally or parenterally. The term "enteral" refers to administration through the gastrointestinal tract, including orally, sublingually, or rectally. Parenteral administration includes intravenous, intradermal, intramuscular, intraperitoneal, subcutaneous, eye, sublingual, intranasal, inhalation, intraspinal, intracerebral, and transdermal (through absorption, for example, through the ducts of the cutaneous glands). The compound or agent may also suitably be administered using reloadable or biodegradable polymer-based products or other products, such as patches and pumps, or formulations that provide sustained, slow, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including a prescribing protocol. For example, in the context of this document, a physician who gives instructions to a patient regarding self-administration of a drug, or regarding administration of a drug by another person, and / or who provides a patient with a drug prescription, is administering the drug to the patient.

Antibodies are typically administered at a concentration in the range of about 0.1 to about 20 mg / kg of patient weight, typically from about 0.5 to about 10 mg / kg, and often from about 1 to about 5 mg / kg. In this regard, it is preferred to use antibodies with a circulating half-life of at least 12 hours, preferably at least 4 days, more preferably up to 21 days. The circulating half-life of chimeric antibodies is expected to be up to 14-21 days. In some cases, it may be advantageous to administer a large loading dose followed by periodic (eg, once a week) maintenance doses during the treatment period. Antibodies can also be delivered using slow release delivery systems, pumps, and other known continuous infusion delivery systems.

The term "approximately" means that for a particular value, an acceptable range of errors should be taken into account, for example, up to 5% or 10%.

Angiogenesis

In one aspect, the present invention provides a pharmaceutical composition according to the present invention for use in the treatment of an angiogenesis-associated disease or disorder.

Angiogenesis is an important cellular event in which vascular endothelial cells proliferate, undergo simplification and reorganization to form new vessels from antecedent vascular networks. There is strong evidence that the development of blood supply is essential for normal and pathological processes of proliferation and inflammation. The vascular compartment is necessary not only for organ development and differentiation during embryogenesis, but also for wound healing, tissue repair, and reproductive function in an adult.

Angiogenesis is also implicated in the pathogenesis of a number of disorders, including, but not limited to, tumors, retinopathies associated with proliferation, age-related macular degeneration, rheumatoid arthritis, and psoriasis. Angiogenesis is extremely important for the growth of most primary tumors and, subsequently, their metastases. Tumors can absorb enough nutrients and oxygen by simple diffusion until they reach 1–2 mm in size, after which a blood supply is required for their further growth. It is believed that this process involves the involvement of adjacent areas of the mature vascular system of the host to initiate the germination of new blood vessels in the form of capillaries that grow in the direction of the tumor mass, and subsequently penetrate into it. In addition, tumor angiogenesis involves recruiting circulating endothelial cell precursors from the bone marrow to promote neovascularization.

Diagnostics

The present invention further discloses methods for diagnosing and determining the prognosis of cancer.

In one aspect, the present invention relates to a method for diagnosing and / or predicting a cancer or infectious disease in a subject, the method comprising the step of determining the level of PVR expression in a biological sample from said subject using at least one antibody as described herein ...

The term "biological sample" encompasses various types of samples obtained from an organism that can be used in a diagnostic or monitoring assay. The term encompasses samples of blood and other fluids of biological origin, solid tissue samples such as biopsy, or tissue or cell cultures derived from and descended from them. In addition, the term can encompass tumor cells or other cells circulating in the bloodstream. The term, in particular, encompasses a clinical preparation, and further includes cells in cell culture, cell supernatants, cell lysates, blood serum, blood plasma, urine, amniotic fluid, biological fluids, including intraocular fluid and vitreous fluid for samples of biological material. eye, and tissue samples. The term also encompasses samples that have been manipulated after preparation, for example, treatment with certain reagents, solubilization or enrichment with certain components.

Determination of the level of PVR expression can be performed using a labeled anti-PVR antibody as described herein. Determination of expression can be performed, for example, using ELISA.

The method of the present invention may further comprise the step of comparing said expression level with a control level.

The following examples are presented to more fully illustrate some embodiments of the present invention. They are in no way to be construed as limiting the scope of the present invention.

Examples of

Experiment procedures

The following examples are given below, which together with the above descriptions illustrate the present invention in a non-limiting manner.

In general, the terminology used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are well known in the art. Other general references related to well-known procedures are provided herein for the convenience of the reader.

Example 1. High level of PVR expression correlates with poor prognosis.

PVR and Nectin-2 are ligands for the TIGIT inhibitory receptor (FIG. 2). The results show that high levels of PVR expression correlated with a poor prognosis for cancer in the case of lung cancer, breast cancer and liposarcoma (Figures 1A-C, respectively). The correlation of PVR expression according to GEO with survival was determined using bioprofiling.de, with the relevant datasets being ID: GSE31210, GSE25055, GSE30929. In addition, using the same assay, it was determined that Nectin-2 expression was primarily a positive marker for survival.

Example 2. Preparation and purification of mAbs to PVR.

In order to obtain antibodies to PVR, a recombinant protein, hPVR-Fc, which combines the extracellular portion of human PVR and the Fc region of a human immunoglobulin G-carrier as an immunogen, was prepared and purified.

BALB / c mice were injected with 50 μg of immunogen in complete Freund's adjuvant, and 2 weeks later - in incomplete Freund's adjuvant. After 2 weeks, serum was screened for antibody titer. The mice with the best response (serum monitored by ELISA for antibody titer to hPVR-Fc) were re-stimulated with the immunogen in PBS. Three days later, spleen cells were harvested, and after erythrocyte lysis, they were fused with SP2 / 0 cells. Cells were plated in 20% RPMI 1640 medium containing hypoxanthine, aminopterin and thymidine to select for hybridomas and screened for mAb production using ELISA. Stable hybridoma cell lines were obtained by fusing SP2 / 0 myeloma cells with spleen cells from an immunized mouse.

Positive results (cell lines secreting antibodies that recognize hPVR-Fc) were further screened to identify a product that would have several distinct characteristics: a) high yield to reduce the cost of antibody production; b) lack of cross-reactivity between mouse and human PVR and several other ligands of immune cell receptors; c) strong binding capacity for native mature human PVR molecules expressed on the surface of living cells (antibodies were selected from a common pool of monoclonal antibodies to hPVR with proven ability to recognize hPVR using various methods, for example, flow cytometry, Western blotting, ELISA ). In fact, human and mouse PVRs have a high level of homology, and it is not easy to obtain a mouse monoclonal antibody that recognizes a human homologue. More importantly, human PVR is extensively glycosylated in its extracellular region. For these reasons, it is not easy to obtain an antibody that recognizes a native protein using common antigens (such as those derived from E. coli).

The authors of the present invention used the immunogen hPVR-Fc, a molecule produced in mammalian cells, which are human embryonic kidney cells 293 (HEK 293T), and purified in native conditions using immunoaffinity chromatography to closely mimic the native protein, human PVR. In conclusion, from the pool of obtained anti-hPVR mAbs, representatives were identified that recognize the native form of human PVR on the surface of living cells, which is a prerequisite for the development of a derivative that will affect the response of a human immune cell during treatment.

Four antibodies to PVR were obtained. Of these, three antibodies were anti-PVR mAb blocking antibodies, namely 5B9 antibody (also called hPVR.09), 7D4 antibody (also called hPVR.01), and 4E5 antibody (also called hPVR.07). All of these antibodies block TIGIT-Ig binding (as illustrated in Figures 3B-D, respectively). A fourth antibody was obtained that does not block TIGIT-Ig binding and was named 2G3 (also called hPVR.17) (Fig. 3A).

Biosensor Biacore ™ T100 surface plasmon resonance (SPR) method (GE Healthcare) was used to determine Koff, Kon and K _D for antibody-hPVR interactions (Table 1).

Table 1. Measurement of antibody affinity using Biacore ™

mAB Affinity 4E5 7.22E ^-10 5B9 1.62E ^-09 7D4 1.93E ^-10

Chimeric monoclonal antibodies containing the human IgG1 heavy chain constant region set forth in SEQ ID NO: 86 (according to GenBank: AAA02914.1) were prepared from the above three antibodies using methods known in the art.

Example 3 Blocking PVR-TIGIT interactions with an anti-PVR mAb increased the killing of human cell lines by NK cells.

Target cells were labeled with [35S] methionine 12 hours prior to analysis. These antibodies were added at a final concentration of 5 μg / ml and incubated with labeled targets (5000 cells / well) for 30 minutes on ice. Assays were performed in RPMI medium in 96-well U-bottomed well plates at 37 ° C for 5 hours. Labeled targets were incubated with NK effector cells at an E: T ratio of 10: 1. After incubation, the plates were centrifuged (1600 rpm, 5 min, 4 ° C) and the supernatants (50 μl) were collected and transferred to Opti-plates (Packard). Added 150 μl of scintillation fluid (Perkin Elmer) and analyzed using a β-counter MicroBeta (Perkin Elmer). The maximum labeling level was determined by adding 100 μl of 0.1 N NaOH to an equal number of targets (5000 / well). Spontaneous release was determined in wells containing only the target cells. The final specific kill rate was calculated as follows: ((radioactivity reading - spontaneous release) / (maximum labeling level - spontaneous release)) * 100 = specific kill. As shown in FIG. 4, culturing NK cells with an anti-PVR mAb 7D4 (also called hPVR.01) increased the killing rate (by a factor of two) by NK cells of the MDA-MB-231 human breast adenocarcinoma cell line.

Example 4 Blocking PVR-TIGIT interactions with anti-PVR mAbs increased the rate of killing by NK cells of human cancer cell lines.

Target cells were labeled with [35S] methionine 12 hours prior to analysis. These antibodies were added at a final concentration of 5 μg / ml and incubated with labeled targets (5000 cells / well) for 30 minutes on ice. The cells were incubated with NK effector cells at an E: T ratio of 10: 1. Assays were performed in RPMI medium in 96-well U-bottomed well plates at 37 ° C for 5 hours. After incubation, the plates were centrifuged (1600 rpm, 5 min, 4 ° C) and the supernatants (50 μl) were collected and transferred to Opti-plates (Packard). Added 150 μl of scintillation fluid (Perkin Elmer) and analyzed using a β-counter MicroBeta (Perkin Elmer). The maximum labeling level was determined by adding 100 μl of 0.1 N NaOH to an equal number of targets (5000 / well). Spontaneous release was determined in wells containing only the target cells. The final specific kill rate was calculated as follows: ((radioactivity reading - spontaneous release) / (maximum labeling level - spontaneous release)) * 100 = specific kill.

As shown in FIG. 5, blocking PVR-TIGIT interactions with anti-PVR mAb 4E5 (also called hPVR.07) increases the rate of killing by NK cells of the HepG2 human cancer cell line (hepatocellular carcinoma). Thus, it is clear that blocking PVR provides an increased rate of target cell killing. Kill was further enhanced by the use of the 4E5 human IgG analog as a chimeric version of the mAb. The kill rate is equivalent to that of the positive control (Erbitux ©).

Example 5 Human tumor cell lines express PVR and Nectin-2.

In order to study the expression of PVR and Nectin-2 on the surface of tumor cells, the expression levels of these proteins in different tumor cell lines were studied by FACS analysis using Ab to PVR-4E5 and Ab to Nectin-2 (clone TX-31), both in concentration 2 μg / ml. As shown in Figures 6A-O, various human tumor cell lines express PVR and Nectin-2. In particular, it is shown that melanoma cells (Figures 6A-E), breast cancer cells (Figures 6F-H), colon and rectal cancer cells (Fig.6I), kidney cancer cells (Fig.6J), lung cancer cells (Fig. 6K), prostate cancer cells (Fig. 6L), brain tumor cells (Fig. 6M), and hepatocellular carcinoma cells (Figures 6N-O) all express PVR and Nectin-2.

Example 6. PVR is the main ligand for TIGIT.

Figures 7A-7C demonstrate that PVR is the main ligand for TIGIT. In particular, HepG2 cells (human hepatocellular carcinoma cells) have been shown to express both PVR and Nectin-2 (Fig. 7A). Culturing HepG2 cells with purified anti-PVR mAb 4E5, also called hPVR.07 (0.15 μg / well), provided almost complete blocking of TIGIT-Ig binding (2 μg / ml) (Fig.7B), regardless of the fact that these cells also express Nectin-2. As shown in FIG. 7C and 7D, it appears that there was no direct recognition of the Nectin-2 mAb to PVR.

Example 7 Binding of mAb Clones to Human and Primate PVR.

As shown in Figures 8A-C, all anti-PVR antibody clones tested bind to human PVR (hPVR) as determined using FACS analysis. Briefly, cells were trypsinized and transferred for staining at a concentration of 2 x 10 ⁵ cells per well. Specified antibodies for 30 min. placed on ice. All antibodies were used at a final concentration of 2 μg / ml. Detection of bound Ab to PVR was performed using IgG-647 to mouse Ig. Mouse IgG1, kappa was used as a negative control.

FIG. 8A illustrates that endogenous hPVR was detected by FACS staining on the cell surface of HepG2 hepatocellular carcinoma. FIG. 8B, the B16 mouse cell line was used. The sequence of mouse PVR is different from that of a human and is not recognized by antibodies to human PVR, as can be seen by the lack of signal (left panel). In the case when the full-length hPVR protein (NP_006496.4, amino acids 1-418) was overexpressed in these cells, all three clones gave an identical signal (right panel), which further confirms the statement that such staining is the result of specific binding of these Ab with human PVR protein.

The amino acid sequence of PVR is conserved among some species. The amino acid conservatism of human PVR was compared with that of African green monkeys in-silico , and the similarity with human PVR was found to be 93%. FACS staining African green monkey Vero cells demonstrated that monkey PVR was efficiently recognized by all three human mAbs (FIG. 8C). It has further been found that such human anti-PVR antibodies do not recognize canine and rodent PVR such as a hamster or mouse. FIG. 9 shows the results of FACS analysis of canine PVR expressing MDCK cells. As can be seen, none of the Ab to human protein gave a positive signal, whereas PVR expression itself was confirmed by a strong TIGIT-Ig signal.

Example 8 Nectin-2 preferably binds with DNAM-1.

10, TIGIT-Fc and DNAM-1-Fc were used at the indicated concentrations to stain cells overexpressing hNectin-2: RPMI-8866 cell lines (Figures 10A and 10B) or B16-hPVR cell lines (Figures 10C and 10D). Detection of the associated fusion protein was performed using APC-labeled anti-human IgG antibody and analyzed by FACS.

In the case of PVR binding, the signal due to TIGIT-Fc was 2-4 times higher than that of DNAM-1-Fc, similar to previously published data (Yu. X et al., 2009, Nat Immunol., 10 (1): 48- 57). Moreover, the binding of hNectin-2 to DNAM-1-Fc was 2-10 times stronger than its binding to TIGIT-Fc, which is not consistent with the results of one work (Yu. X et al 2009), but is confirmed by another study (Zhu Y et al., 2016, J Exp Med., 8; 213 (2): 167-76). Together, these results indicate that DNAM-1 is the preferred receptor for Nectin-2 binding. Accordingly, blocking PVR will prevent transmission of an inhibitory signal from TIGIT, while allowing transmission of a costimulatory signal from DNAM-1. DNAM-1 mediates cell adhesion to other cells bearing its ligands.

Example 9 Anti-PVR Antibodies Provide Enhanced T-Cell Proliferation.

To test the effect of anti-PVR mAbs on T cell proliferation, human peripheral blood mononuclear cells (PBMCs) were stained with carboxyfluorescein succinimidyl ester (CFSE) and incubated with target cells in the presence of antibodies at a concentration of 4 μg / ml. Dilution of CFSE was measured for CD45-positive cells after 5-9 days of culture. As shown in FIG. 11, the activity of 5B9 against PVR exceeds that of antibodies against PD-1 and CTLA4 as a separate agent. In addition, the chimeric clone 4E5hIgG1 to PVR with a constant region of human IgG1 was superior to its analog in the mouse. The combined effect of anti-PVR mAbs and other antibodies, which were found to enhance T cell proliferation, was further examined. Human PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and incubated with target cells in the presence of 4 μg / ml antibodies. Dilution of CFSE was measured for CD45-positive cells after 5-9 days of culture. The results show that the proliferative activity of 5B9 against PVR when combined with either an anti-PD-1 antibody or a CTLA-4 antibody is superior to that with a combination of PD-1 and CTLA4 (FIG. 12). In addition, the activity of the combination of 4E5 against PVR and CTLA-4 is similar to that of the combination of PD-1 and CTLA4. Further, the specific induction of CD8 was studied. As shown in FIG. 13, the activity of anti-PVR antibodies against the proliferation of CD8 T cells exceeds that of PD1. In addition, the induction activity of the anti-PVR antibody 5B9 exceeds that of CTLA1. For different antibodies, the ratio of CD8 / CD4 cell proliferation was assessed (Fig. 14). 5B9 to PVR had the highest CD8 / CD4 ratio. The DNA sequence of the variable region of the heavy and light chains was used to construct chimeric antibodies containing the constant domains of the human IgG1 isotype and the constant domain of the human IgG kappa light (CL) kappa chain. T cell proliferation induced by chimeric analogs of murine and human antibodies was measured by CFSE assay. Presented in Table 2, the numbers represent the relative level of proliferation compared to the control.

Table 2. Summary of the effect of anti-PVR antibodies on T cell proliferation.

Clone name Fc type T cell proliferation effect (CFSE) Effect on T cell proliferation, cell number 4E5 IgG1 mouse 198% 320% IgG1 human 275% 353% 5B9 IgG1 mouse 300% 410% IgG1 human 270% 364% 7D4 IgG1 mouse 141% 260% IgG1 human 280% 360%

Next, the effect of PVR antibodies on NK cell degranulation was studied. During degranulation, cytolytic granules are released from NK cells, and the membrane protein-1 (LAMP-1, CD107a) associated with lysosomes, which is present on the surface of cytolytic granules, is transported to the cell surface and becomes available for antibody binding. This marker allows the identification of activated NK cells. NK cells were incubated with 5 different target cells, together with anti-PVR antibodies (mouse antibodies and their chimeric analogs with human sequences). Degranulation was assessed using anti-CD107a antibodies.

Table 3. Effect of anti-PVR antibodies on NK cell degranulation activity .

Target cancer cells Clone Fc type MDA-MB-231 HepG2 MV-411 Mel-624 * A549 4E5 IgG1 mouse 125% 150% 150% 120% - IgG1 human 305% 260% 160% 145% 260% 5B9 IgG1 mouse 132% 160% 150% 120% - IgG1 human 300% 260% 220% 145% 240% 7D4 IgG1 mouse 124% 160% 150% 115% - IgG1 human 220% 200% 130% 120% 165%

* the expression level of hPVR in Mel 624 is low, hence the relative effect is low.

As shown in Table 3 and FIG. 15, all antibodies were shown to be active against NK degranulation, where the chimeric antibodies were significantly more active than their corresponding mouse antibodies.

Example 10 Antibodies to PVR provide a decrease in tumor cell survival in the absence of immune cells.

The viability of A549, U373, HCT116 and Mel-624 cells was studied using the MTT cell viability assay in the presence of 50 micrograms / ml of different anti-PVR mAbs for 24 hours. As shown in Figures 16A-16D and Table 4, PVR blocking with mAb 5B9 resulted in a significant reduction in viability of 20-40% compared to mIgG.

Table 4. Effect of anti-PVR antibodies on tumor cell survival.

Percentage of dead cells over a 24 hour period among multiple target cell lines compared to cells treated with mIgG.

mAb MDA-MB-231 HCT-116 Mel-624 A549 4E5 12% twenty% - 25% 5B9 twenty% 22% 32% 25% 7D4 17% 27% 12% 25%

Example 11 In-Vivo Effect of Anti-PVR Antibodies on Human Tumor in Humanized Mouse Model - Transient Humanization.

The antitumor efficacy of antibodies was studied in vivo. To evaluate the effectiveness of the antibodies described herein in inhibiting human cancer, the antibody was tested in a model combining both tumors and human lymphocytes. NOD scid gamma (NSG) mice were inoculated with hPBMC to restore immunocompetence and injected with human cancer cells. At a given time point, mice with a defined tumor size were treated with an anti-human PVR antibody according to the present invention, administered in multiple doses at different time points after tumor injection. The same experiments were performed with chimeric anti-PVR antibodies. Tumor growth curves and body weight were measured 3x / week, and after sacrifice of mice, detailed phenotypic analysis of TIL and immune cell populations in different organs was performed.

A similar model with tumor cell lines in huNSG mice inoculated with PBMC was created according to Gupta P., Oncoimmunology. 2015 Mar 6; 4 (2): e981449.

Example 12 In-Vivo Effect of Anti-PVR Antibodies on Human Tumor in Humanized Mouse Model - Long-Term Humanization.

To evaluate the effectiveness of anti-PVR antibodies in inhibiting human cancer, the antibody was tested in a model combining both tumors and human-derived lymphocytes. Newborn NOD scid gamma (NSG) pups were irradiated and inoculated with CD34 + HSCs to restore immunocompetence. 1-2 weeks after determining the replacement of immune cells, the mice were injected with human cancer cells, and at a given time point / with a certain tumor size were treated with an antibody to human PVR in accordance with the present invention, administered in several doses at different time points. The same experiments were performed with humanized anti-PVR antibodies. Tumor growth curves and body weight were measured 3x / week. After sacrificing the mice, a detailed phenotypic analysis of TIL and populations of immune cells in different organs was performed.

A similar model used to study the tumor inhibitory activity of the antibodies of the present invention was created at The Jackson Laboratory (http://immune-checkpoint.com/wp-content/uploads/sites/24/2015/01/ Day-1-15.45-Rick-Huntress.pdf).

Example 13. Induction of IFNγ secretion.

To test the effect of anti-PVR mAbs on cytokine secretion, human peripheral blood mononuclear cells (PBMCs) from 2 healthy donors were incubated with target cells in the presence of antibodies (mIgG, 5B9mIgG, an anti-CTL-4 antibody called ipilimumab) at concentrations of 1 and 0.1 μg / ml. IFNy levels were measured after 6 days of culture. Significant induction of IFNγ by the anti-PVR antibody 5B9 was observed (P = 7.34548E ^-11 for 1 μg / ml and 2.73179E ^-08 for 0.1 μg / ml).

IFNγ secretion is a key component of anti-tumor immunity, and induction of IFNγ secretion by anti-PVR mAb indicates a potential additional anti-cancer effect of these compounds in cancer treatment.

The foregoing description of specific embodiments has sufficiently disclosed the general nature of the present invention so that others can, using their existing knowledge, easily modify and / or adapt such specific embodiments for different applications without undue experimentation or deviation from the general concept, and therefore such adaptations and modifications are to be understood, and as intended, in light and in a series of equivalents of the disclosed embodiments. It should be understood that the language or terminology used in this document is for purposes of description and not limitation.

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LIST OF SEQUENCES

<110> YUSSUM RISERCH DEVELOPMENT COMPANY OF ZE HIBRU

UNIVERSITY OF JERUSALEM LTD.

UNIVERSITY OF RIEKA FECALTI OF MEDISIN

<120> ANTIBODIES SPECIFIC TO THE HUMAN POLIOVIRUS RECEPTOR (PVR)

<130> Yissum / 0137 PCT

<150> US 62/301727

<151> 2016-03-01

<150> US 62/364924

<151> 2016-07-21

<160> 86

<170> PatentIn version 3.5

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<213> Mus musculus

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ccagggaaag ggctagaatg gattggagaa attcatccag atagcagtaa gataaactat 180

acgccatctc aaaaggataa attcatcatc tccagagaca acgccaaaaa tacgctgttc 240

ctgcaaatga gcaaagtgag atttgaggac acagcccttt atttctgtgc aagacctgat 300

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Gly Glu Ile His Pro Asp Ser Ser Lys Ile Asn Tyr Thr Pro Ser Gln

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Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

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Leu Gln Met Ser Lys Val Arg Phe Glu Asp Thr Ala Leu Tyr Phe Cys

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Ala Arg Pro Asp Gly Asn Tyr Asn Ala Leu Asp Tyr Trp Gly Gln Gly

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Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr

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Val Asp

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ggcacactgg agtccctgat cgcttcacag gcagtgaatc tgggacagat ttcactctca 240

ccattagtga tgtgcaatct gaagacttgg caaattattt ctgtcagcaa tatagcaggt 300

atccgtacac gttcggaggg gggaccaagc tggaaataaa acgggccgat gctgcaccaa 360

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Ala Thr Gly Val His Ser Asp Ile Val Met Thr Gln Ser His Lys Phe

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Gln Asp Val Gly Thr Ala Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln

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Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val

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Pro Asp Arg Phe Thr Gly Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr

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Ile Ser Asp Val Gln Ser Glu Asp Leu Ala Asn Tyr Phe Cys Gln Gln

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Tyr Ser Arg Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

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Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Val Asp

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Lys Ala Ser Gln Asp Val Gly Thr Ala Val Thr

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Trp Ala Ser Thr Arg His Thr

15

<210> 15

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<210> 16

<211> 9

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Gln Gln Tyr Ser Arg Tyr Pro Tyr Thr

15

<210> 17

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<213> Mus musculus

<400> 17

gaattcgagg tgaagctgca ggagtctgga cctgagctgg tgaagcctgg ggcttcagtg 60

aagatttcct gcaagacttc tggatacact ttcactgaat acaccatgca ctgggtgagg 120

cagagccatg gagagagcct tgagtggatt ggaggtattg atcctaacaa tggtggtact 180

aactacaacc agaacttcaa gggcaaggcc acattgactg tagacaagtc ctccagcaca 240

gcctacatgg agctccgcag cctgacatct gaggattctg cggtctatta ctgtgcagga 300

gtgattcctc tggagtactg ggggcaagga acctcagtca ccgtctcctc agccaaaacg 360

acacccccat ctgtctatgt cgaccatatg ggagagctcc caacgcgttg gatgcatagc 420

ttgagtattc tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct 480

gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccg 526

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Glu Phe Glu Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro

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Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr

20 25 30

Glu Tyr Thr Met His Trp Val Arg Gln Ser His Gly Glu Ser Leu Glu

35 40 45

Trp Ile Gly Gly Ile Asp Pro Asn Asn Gly Gly Thr Asn Tyr Asn Gln

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Asn Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr

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Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

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Tyr Cys Ala Gly Val Ile Pro Leu Glu Tyr Trp Gly Gln Gly Thr Ser

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Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Val Asp

115 120 125

His Met Gly Glu Leu Pro Thr Arg Trp Met His Ser Leu Ser Ile Leu

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<213> Mus musculus

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ggatacactt tcactgaata caccatgcac 30

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Gly Tyr Thr Phe Thr Glu Tyr Thr Met His

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<211> 51

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<210> 22

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Gly Ile Asp Pro Asn Asn Gly Gly Thr Asn Tyr Asn Gln Asn Phe Lys

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<212> DNA

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<210> 24

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<400> 24

Val Ile Pro Leu Glu Tyr

15

<210> 25

<211> 321

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<400> 25

gatattgtga tgacacagtc tcaaaaattc atgtccacat cagtaggaga cagagtcagc 60

gtcacctgca aggccagtca gaatgtgtat actaatgtag cctggtatca acagaaacca 120

gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagggg agtccctgat 180

cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagacttgg cagactattt ctgtcagcaa tataacagct atcctctcgc gttcggctcg 300

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<212> PROTEIN

<213> Mus musculus

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Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

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Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Tyr Thr Asn

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

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Tyr Ser Ala Ser Tyr Arg Tyr Arg Gly Val Pro Asp Arg Phe Thr Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

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Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

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Ala Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

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Pro Thr Val Ser Val Asp

115

<210> 27

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<210> 29

<211> 21

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<400> 29

tcggcatcct accggtacag g 21

<210> 30

<211> 7

<212> PROTEIN

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<400> 30

Ser Ala Ser Tyr Arg Tyr Arg

15

<210> 31

<211> 27

<212> DNA

<213> Mus musculus

<400> 31

cagcaatata acagctatcc tctcgcg 27

<210> 32

<211> 9

<212> PROTEIN

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<400> 32

Gln Gln Tyr Asn Ser Tyr Pro Leu Ala

15

<210> 33

<211> 345

<212> DNA

<213> Mus musculus

<400> 33

caactgcagc agtctggagc tgagctgatg aagcctgggg cctcagtgaa gatttcctgc 60

aaggctactg gctacacatt cagtaactac tggattgagt ggataaaaca gaggcctgga 120

catggccttg agtggattgg agagattttt cctggaagtg gtcgtattaa cttcaatgag 180

aagttcaagg gcaaggccac attcactgca gacacatcct ccgacacaac ctacatgcaa 240

ctcagcagcc tgacatctgc ggactctgcc gtctattact gtgcaagaac gaagatctat 300

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Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala Ser Val

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Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Glu

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Ile Phe Pro Gly Ser Gly Arg Ile Asn Phe Asn Glu Lys Phe Lys Gly

50 55 60

Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asp Thr Thr Tyr Met Gln

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Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Thr Lys Ile Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val

115

<210> 35

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ggctacacat tcagtaacta ctggattgag 30

<210> 36

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<212> PROTEIN

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Gly Tyr Thr Phe Ser Asn Tyr Trp Ile Glu

1 5 10

<210> 37

<211> 51

<212> DNA

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<210> 38

<211> 16

<212> PROTEIN

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<400> 38

Glu Ile Phe Pro Gly Ser Gly Arg Ile Asn Phe Asn Glu Lys Phe Lys

1 5 10 15

<210> 39

<211> 30

<212> DNA

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acgaagatct atggtaactc ctttgactac 30

<210> 40

<211> 10

<212> PROTEIN

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<400> 40

Thr Lys Ile Tyr Gly Asn Ser Phe Asp Tyr

1 5 10

<210> 41

<211> 324

<212> DNA

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<400> 41

gacatcgtga tgactcagtc tcacaaattc atgtccacat cagtaggaga cagggtcaac 60

atcacctgca aggccagtca ggatgtgggt actgctgtgg tctggtatca acagaaacca 120

ggacaatctc ctaaattatt gatttactgg gcatccagtc ggcacaatgg agtccctgat 180

cgcttcacag gcagtggatc tgggacagat ttcactctca cgattagtaa tgtgcagtct 240

gaagacttgt cagattattt ctgtcagcaa tatagcaggt atccactcac attcggggct 300

gggaccaagc tggagctgaa acgt 324

<210> 42

<211> 108

<212> PROTEIN

<213> Mus musculus

<400> 42

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Ser Arg His Asn Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ser Asp Tyr Phe Cys Gln Gln Tyr Ser Arg Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210> 43

<211> 33

<212> DNA

<213> Mus musculus

<400> 43

aaggccagtc aggatgtggg tactgctgtg gtc 33

<210> 44

<211> 11

<212> PROTEIN

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Lys Ala Ser Gln Asp Val Gly Thr Ala Val Val

1 5 10

<210> 45

<211> 21

<212> DNA

<213> Mus musculus

<400> 45

tgggcatcca gtcggcacaa t 21

<210> 46

<211> 7

<212> PROTEIN

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<400> 46

Trp Ala Ser Ser Arg His Asn

15

<210> 47

<211> 27

<212> DNA

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cagcaatata gcaggtatcc actcaca 27

<210> 48

<211> 9

<212> PROTEIN

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<400> 48

Gln Gln Tyr Ser Arg Tyr Pro Leu Thr

15

<210> 49

<211> 107

<212> PROTEIN

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<220>

<223> Mutated Chain

<400> 49

Asp Ile Met Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Ser Arg His Ala Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ser Asp Tyr Phe Cys Gln Gln Tyr Ser Arg Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 50

<211> 107

<212> PROTEIN

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<220>

<223> Mutated Chain

<400> 50

Asp Ile Met Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Ser Arg His Arg Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ser Asp Tyr Phe Cys Gln Gln Tyr Ser Arg Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 51

<211> 107

<212> PROTEIN

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<220>

<223> Mutated Chain

<400> 51

Asp Ile Met Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Ser Arg His Asp Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ser Asp Tyr Phe Cys Gln Gln Tyr Ser Arg Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 52

<211> 107

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<220>

<223> Mutated Chain

<400> 52

Asp Ile Met Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Ser Arg His Glu Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ser Asp Tyr Phe Cys Gln Gln Tyr Ser Arg Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 53

<211> 107

<212> PROTEIN

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<220>

<223> Mutated Chain

<400> 53

Asp Ile Met Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Ser Arg His Pro Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ser Asp Tyr Phe Cys Gln Gln Tyr Ser Arg Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 54

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutated Chain

<400> 54

Asp Ile Met Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Ser Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ser Asp Tyr Phe Cys Gln Gln Tyr Ser Arg Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 55

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutated CDR

<220>

<221> MISC_FEATURE

<222> (7) .. (7)

<223> Xaa is an amino acid residue selected from: Ala, Arg, Asp, Glu, Pro and Thr

<400> 55

Trp Ala Ser Ser Arg His Xaa

15

<210> 56

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutated CDR

<400> 56

Trp Ala Ser Ser Arg His Ala

15

<210> 57

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutated CDR

<400> 57

Trp Ala Ser Ser Arg His Arg

15

<210> 58

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutated CDR

<400> 58

Trp Ala Ser Ser Arg His Asp

15

<210> 59

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutated CDR

<400> 59

Trp Ala Ser Ser Arg His Glu

15

<210> 60

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutated CDR

<400> 60

Trp Ala Ser Ser Arg His Pro

15

<210> 61

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mutated CDR

<400> 61

Trp Ala Ser Ser Arg His Thr

15

<210> 62

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Mutated polynucleotide

<400> 62

gacatcatga tgacccagtc tcacaaattc atgtctacat ctgtaggaga cagagtcaac 60

atcacttgca aggcgagtca ggatgtgggt actgctgtgg tctggtatca gcagaaacca 120

gggcaatctc ctaagttgct gatctattgg gcatccagtc ggcacgctgg ggtcccagat 180

aggttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagatttgt cagattattt ctgccaacag tatagcagat accctctcac attcggggct 300

gggaccaagc tggagctcaa a 321

<210> 63

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Mutated polynucleotide

<400> 63

gacatcatga tgacccagtc tcacaaattc atgtctacat ctgtaggaga cagagtcaac 60

atcacttgca aggcgagtca ggatgtgggt actgctgtgg tctggtatca gcagaaacca 120

gggcaatctc ctaagttgct gatctattgg gcatccagtc ggcacagagg ggtcccagat 180

aggttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagatttgt cagattattt ctgccaacag tatagcagat accctctcac attcggggct 300

gggaccaagc tggagctcaa a 321

<210> 64

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Mutated polynucleotide

<400> 64

gacatcatga tgacccagtc tcacaaattc atgtctacat ctgtaggaga cagagtcaac 60

atcacttgca aggcgagtca ggatgtgggt actgctgtgg tctggtatca gcagaaacca 120

gggcaatctc ctaagttgct gatctattgg gcatccagtc ggcacgatgg ggtcccagat 180

aggttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagatttgt cagattattt ctgccaacag tatagcagat accctctcac attcggggct 300

gggaccaagc tggagctcaa a 321

<210> 65

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Mutated polynucleotide

<400> 65

gacatcatga tgacccagtc tcacaaattc atgtctacat ctgtaggaga cagagtcaac 60

atcacttgca aggcgagtca ggatgtgggt actgctgtgg tctggtatca gcagaaacca 120

gggcaatctc ctaagttgct gatctattgg gcatccagtc ggcacgaggg ggtcccagat 180

aggttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagatttgt cagattattt ctgccaacag tatagcagat accctctcac attcggggct 300

gggaccaagc tggagctcaa a 321

<210> 66

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Mutated polynucleotide

<400> 66

gacatcatga tgacccagtc tcacaaattc atgtctacat ctgtaggaga cagagtcaac 60

atcacttgca aggcgagtca ggatgtgggt actgctgtgg tctggtatca gcagaaacca 120

gggcaatctc ctaagttgct gatctattgg gcatccagtc ggcaccctgg ggtcccagat 180

aggttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagatttgt cagattattt ctgccaacag tatagcagat accctctcac attcggggct 300

gggaccaagc tggagctcaa a 321

<210> 67

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Mutated polynucleotide

<400> 67

gacatcatga tgacccagtc tcacaaattc atgtctacat ctgtaggaga cagagtcaac 60

atcacttgca aggcgagtca ggatgtgggt actgctgtgg tctggtatca gcagaaacca 120

gggcaatctc ctaagttgct gatctattgg gcatccagtc ggcacactgg ggtcccagat 180

aggttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagatttgt cagattattt ctgccaacag tatagcagat accctctcac attcggggct 300

gggaccaagc tggagctcaa a 321

<210> 68

<211> 356

<212> DNA

<213> Mus musculus

<400> 68

gaggtgaagc ttctcgagtc tggaggtggc ctggtgcagc ctggaggatc cctgaaactc 60

tcctgtgcag cctcaggatt cgattttagt agatactgga tgacttgggt ccggcaggct 120

ccagggaaag ggctagaatg gattggagaa attcatccag atagcagtaa gataaactat 180

acgccatctc aaaaggataa attcatcatc tccagagaca acgccaaaaa tacgctgttc 240

ctgcaaatga gcaaagtgag atttgaggac acagcccttt atttctgtgc aagacctgat 300

ggtaactaca atgctctgga ctactggggt caaggaacct cagtcaccgt ctcctc 356

<210> 69

<211> 119

<212> PROTEIN

<213> Mus musculus

<400> 69

Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr

20 25 30

Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile His Pro Asp Ser Ser Lys Ile Asn Tyr Thr Pro Ser Gln

50 55 60

Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Ser Lys Val Arg Phe Glu Asp Thr Ala Leu Tyr Phe Cys

85 90 95

Ala Arg Pro Asp Gly Asn Tyr Asn Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 70

<211> 321

<212> DNA

<213> Mus musculus

<400> 70

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtgggaga cagggtcagc 60

atcacctgca aggccagtca ggatgtgggt actgctgtaa cctggtatca acagaaacca 120

ggacaatctc ctaaactact gatttactgg gcatccaccc ggcacactgg agtccctgat 180

cgcttcacag gcagtgaatc tgggacagat ttcactctca ccattagtga tgtgcaatct 240

gaagacttgg caaattattt ctgtcagcaa tatagcaggt atccgtacac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210> 71

<211> 107

<212> PROTEIN

<213> Mus musculus

<400> 71

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asp Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asn Tyr Phe Cys Gln Gln Tyr Ser Arg Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 72

<211> 345

<212> DNA

<213> Mus musculus

<400> 72

gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatt 60

tcctgcaaga cttctggata cactttcact gaatacacca tgcactgggt gaagcagagc 120

catggagaga gccttgagtg gattggaggt attgatccta acaatggtgg tactaactac 180

aaccagaact tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240

atggagctcc gcagcctgac atctgaggat tctgcggtct attactgtgc aggagtgatt 300

cctctggagt actgggggca aggaacctca gtcaccgtct cctca 345

<210> 73

<211> 115

<212> PROTEIN

<213> Mus musculus

<400> 73

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Met His Trp Val Lys Gln Ser His Gly Glu Ser Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asp Pro Asn Asn Gly Gly Thr Asn Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Val Ile Pro Leu Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr

100 105 110

Val Ser Ser

115

<210> 74

<211> 321

<212> DNA

<213> Mus musculus

<400> 74

gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagagtcagc 60

gtcacctgca aggccagtca gaatgtgtat actaatgtag cctggtatca acagaaacca 120

gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagggg agtccctgat 180

cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagacttgg cagactattt ctgtcagcaa tataacagct atcctctcgc gttcggctcg 300

gggacaaagt tggaaataaa a 321

<210> 75

<211> 107

<212> PROTEIN

<213> Mus musculus

<400> 75

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Tyr Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Arg Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Ala Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 76

<211> 357

<212> DNA

<213> Mus musculus

<400> 76

caggttcagt tgcagcagtc tggagctgag ctgatgaagc ctggggcctc agtgaagatt 60

tcctgcaagg ctactggcta cacattcagt aactactgga ttgagtggat aaaacagagg 120

cctggacatg gccttgagtg gattggagag atttttcctg gaagtggtcg tattaacttc 180

aatgagaagt tcaagggcaa ggccacattc actgcagaca catcctccga cacaacctac 240

atgcaactca gcagcctgac atctgcggac tctgccgtct attactgtgc aagaacgaag 300

atctatggta actcctttga ctactggggc caaggcacca ctctcacagt ctcccca 357

<210> 77

<211> 119

<212> PROTEIN

<213> Mus musculus

<400> 77

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Phe Pro Gly Ser Gly Arg Ile Asn Phe Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asp Thr Thr Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Lys Ile Tyr Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Pro

115

<210> 78

<211> 321

<212> DNA

<213> Mus musculus

<400> 78

gacattatga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcaac 60

atcacctgca aggccagtca ggatgtgggt actgctgtgg tctggtatca acagaaacca 120

ggacaatctc ctaaattatt gatttactgg gcatccagtc ggcacaatgg agtccctgat 180

cgcttcacag gcagtggatc tgggacagat ttcactctca cgattagtaa tgtgcagtct 240

gaagacttgt cagattattt ctgtcagcaa tatagcaggt atccactcac attcggggct 300

gggaccaagc tggagctgaa a 321

<210> 79

<211> 107

<212> PROTEIN

<213> Mus musculus

<400> 79

Asp Ile Met Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Ser Arg His Asn Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ser Asp Tyr Phe Cys Gln Gln Tyr Ser Arg Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 80

<211> 5

<212> PROTEIN

<213> Mus musculus

<400> 80

Arg Tyr Trp Met Thr

15

<210> 81

<211> 17

<212> PROTEIN

<213> Mus musculus

<400> 81

Glu Ile His Pro Asp Ser Ser Lys Ile Asn Tyr Thr Pro Ser Gln Lys

1 5 10 15

Asp

<210> 82

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 82

Pro Asp Gly Asn Tyr Asn Ala Leu Asp Tyr

1 5 10

<210> 83

<211> 5

<212> PROTEIN

<213> Mus musculus

<400> 83

Glu Tyr Thr Met His

15

<210> 84

<211> 6

<212> PROTEIN

<213> Mus musculus

<400> 84

Ser Asn Tyr Trp Ile Glu

15

<210> 85

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 85

Lys Ala Ser Gln Asp Val Gly Thr Ala Val

1 5 10

<210> 86

<211> 329

<212> PROTEIN

<213> Homo sapiens

<400> 86

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

1 5 10 15

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

20 25 30

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

35 40 45

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

50 55 60

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

65 70 75 80

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

85 90 95

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

100 105 110

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

115 120 125

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

130 135 140

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

145 150 155 160

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

165 170 175

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

180 185 190

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

195 200 205

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

210 215 220

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

225 230 235 240

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

245 250 255

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

260 265 270

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

275 280 285

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

290 295 300

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

305 310 315 320

Lys Ser Leu Ser Leu Ser Pro Gly Lys

325

<---

Claims (34)

1. An isolated monoclonal antibody that specifically binds to the human poliovirus receptor (PVR), or an antibody fragment thereof containing at least an antigen-binding region, containing a set of CDRs selected from the group consisting of: i. a CDR set of six CDRs, where: CDR1 HC is selected from GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84); CDR2 HC is EIFPGSGRINFNEKFKG (SEQ ID NO: 38); CDR3 HC is TKIYGNSFDY (SEQ ID NO: 40); CDR1 LC selected from KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85); CDR2 LC is selected from the group consisting of: WASSRHN (SEQ ID NO: 46), WASSRHA (SEQ ID NO: 56), WASSRHR (SEQ ID NO: 57), WASSRHD (SEQ ID NO: 58), WASSRHE (SEQ ID NO : 59), WASSRHP (SEQ ID NO: 60) and WASSRHT (SEQ ID NO: 61); and CDR3 LC is QQYSRYPLT (SEQ ID NO: 48); ii. a set of CDRs of six CDRs, where: CDR1 HC is selected from GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); CDR2 HC is selected from EIHPDSSKINYTPSQ (SEQ ID NO: 6) and EIHPDSSKINYTPSQKD (SEQ ID NO: 81); CDR3 HC is selected from PDGNYNALDYW (SEQ ID NO: 8) and PDGNYNALDY (SEQ ID NO: 82); CDR1 LC is KASQDVGTAVT (SEQ ID NO: 12); CDR2 LC is WASTRHT (SEQ ID NO: 14); and CDR3 LC is QQYSRYPYT (SEQ ID NO: 16); and iii. a CDR set of six CDRs, where: CDR1 HC is selected from GYTFTEYTMH (SEQ ID NO: 20) and EYTMH (SEQ ID NO: 83); CDR2 HC is GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); CDR3 HC is VIPLEY (SEQ ID NO: 24); CDR1 LC is KASQNVYTNVA (SEQ ID NO: 28); CDR2 LC is SASYRYR (SEQ ID NO: 30); and CDR3 LC is QQYNSYPLA (SEQ ID NO: 32). 2. An isolated monoclonal antibody or antibody fragment according to claim 1, wherein the HC CDR1 contains the sequence SNYWIE (SEQ ID NO: 84); CDR2 HC contains the sequence set forth in EIFPGSGRINFNEKFKG (SEQ ID NO: 38); and CDR3 HC contains the sequence: TKIYGNSFDY (SEQ ID NO: 40). 3. An isolated monoclonal antibody or antibody fragment according to claim 1 or 2, wherein the CDR1 LC contains the sequence KASQDVGTAV (SEQ ID NO: 85); CDR2 LC contains the sequence: WASSRHN (SEQ ID NO: 46); and CDR3 LC contains the sequence: QQYSRYPLT (SEQ ID NO: 48). 4. Isolated monoclonal antibody or antibody fragment according to any one of claims. 1-3, where the HC CDR1 sequence is selected from the group consisting of GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84); the CDR2 HC sequence consists of the sequence EIFPGSGRINFNEKFKG (SEQ ID NO: 38); CDR3 HC consists of the sequence: TKIYGNSFDY (SEQ ID NO: 40); the CDR1 LC sequence is selected from the group consisting of KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85); CDR2 LC consists of the sequence: WASSRHN (SEQ ID NO: 46); and CDR3 LC consists of the sequence: QQYSRYPLT (SEQ ID NO: 48). 5. Isolated monoclonal antibody or antibody fragment according to any one of claims. 1-4 comprising a heavy chain variable region selected from SEQ ID NO: 34 and SEQ ID NO: 77, or an analog having at least 95% sequence similarity to said heavy chain variable region sequence. 6. Isolated monoclonal antibody or antibody fragment according to any one of claims. 1-5, containing a heavy chain and a light chain, where the heavy chain contains SEQ ID NO: 77 and the light chain contains SEQ ID NO: 79. 7. An isolated monoclonal antibody or antibody fragment according to claim 1, wherein the HC CDR1 sequence is selected from the group consisting of GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84); the CDR2 HC sequence consists of EIFPGSGRINFNEKFKG (SEQ ID NO: 38); CDR3 HC consists of the sequence: TKIYGNSFDY (SEQ ID NO: 40); the CDR1 LC sequence is selected from the group consisting of KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85); CDR2 LC sequence is selected from the group consisting of WASSRHN (SEQ ID NO: 46), WASSRHA (SEQ ID NO: 56), WASSRHR (SEQ ID NO: 57), WASSRHD (SEQ ID NO: 58), WASSRHE (SEQ ID NO : 59), WASSRHP (SEQ ID NO: 60) and WASSRHT (SEQ ID NO: 61); and CDR3 LC consists of the sequence: QQYSRYPLT (SEQ ID NO: 48). 8. An isolated monoclonal antibody or antibody fragment according to claim 1, wherein the HC CDR1 contains a sequence selected from the group consisting of GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); CDR2 HC contains the sequence EIHPDSSKINYTPSQ (SEQ ID NO: 6); and CDR3 HC contains the sequence PDGNYNALDY (SEQ ID NO: 82). 9. An isolated monoclonal antibody or antibody fragment according to any one of claims 1 or 8, wherein the LC CDR1 contains the sequence: KASQDVGTAVT (SEQ ID NO: 12); CDR2 LC contains the sequence: WASTRHT (SEQ ID NO: 14); and CDR3 LC contains the sequence: QQYSRYPYT (SEQ ID NO: 16). 10. An isolated monoclonal antibody or antibody fragment according to claim 1, wherein the HC CDR1 sequence is selected from the group consisting of GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); the CDR2 HC sequence is selected from the group consisting of EIHPDSSKINYTPSQ (SEQ ID NO: 6) and EIHPDSSKINYTPSQKD (SEQ ID NO: 81); the CDR3 HC sequence is selected from the group consisting of PDGNYNALDYW (SEQ ID NO: 8) and PDGNYNALDY (SEQ ID NO: 82); CDR1 LC consists of the sequence: KASQDVGTAVT (SEQ ID NO: 12); CDR2 LC consists of the sequence: WASTRHT (SEQ ID NO: 14); and CDR3 LC consists of the sequence: QQYSRYPYT (SEQ ID NO: 16). 11. The isolated monoclonal antibody or antibody fragment according to any one of claims 1 and claims. 8-10, containing a heavy chain and a light chain, where the heavy chain contains SEQ ID NO: 69 and the light chain contains SEQ ID NO: 71. 12. An isolated monoclonal antibody or antibody fragment according to claim 1, wherein the HC CDR1 contains the sequence EYTMH (SEQ ID NO: 83); CDR2 HC contains the sequence GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); and CDR3 HC contains the sequence: VIPLEY (SEQ ID NO: 24). 13. An isolated monoclonal antibody or antibody fragment according to any one of claims 1 or 12, wherein the LC CDR1 contains the sequence: KASQNVYTNVA (SEQ ID NO: 28); CDR2 LC contains the sequence: SASYRYR (SEQ ID NO: 30); and CDR3 LC contains the sequence: QQYNSYPLA (SEQ ID NO: 32). 14. An isolated monoclonal antibody or antibody fragment according to claim 1, wherein the HC CDR1 sequence is selected from the group consisting of GYTFTEYTMH (SEQ ID NO: 20) and EYTMH (SEQ ID NO: 83); the CDR2 HC sequence consists of GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); CDR3 HC consists of the sequence: VIPLEY (SEQ ID NO: 24); LC CDR1 consists of the sequence: KASQNVYTNVA (SEQ ID NO: 28); CDR2 LC consists of the sequence: SASYRYR (SEQ ID NO: 30); and CDR3 LC consists of the sequence: QQYNSYPLA (SEQ ID NO: 32). 15. An isolated monoclonal antibody or antibody fragment according to any one of claims 1 and 3. 12-14, containing a heavy chain and a light chain, where the heavy chain contains SEQ ID NO: 73 and the light chain contains SEQ ID NO: 75. 16. Isolated monoclonal antibody according to any one of paragraphs. 1-15, capable of inhibiting the binding of PVR to T-cell immunoreceptor with domains Ig and ITIM (TIGIT). 17. Polynucleotide encoding the sequence of the variable region of the heavy chain of the monoclonal antibody according to any one of paragraphs. 1-15. 18. The polynucleotide of claim 17, encoding a heavy chain variable region of a monoclonal antibody, wherein the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 68, SEQ ID NO: 17, SEQ ID NO: 72 , SEQ ID NO: 33 and SEQ ID NO: 76, or a variant thereof, having at least 90% identity with the indicated sequences. 19. A polynucleotide encoding the sequence of the variable region of the light chain of a monoclonal antibody according to any one of claims. 1-15. 20. The polynucleotide of claim 19, encoding the variable region of the light chain of a monoclonal antibody, wherein the polynucleotide comprises a sequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 70, SEQ ID NO: 25, SEQ ID NO: 74, SEQ ID NO: 41 and SEQ ID NO: 78, or a variant thereof, having at least 90% identity with said sequences. 21. Plasmid for producing a monoclonal antibody that specifically binds to the human poliovirus receptor (PVR), or an antibody fragment thereof, containing at least an antigen-binding site, wherein the plasmid comprises a polynucleotide according to claim 18 and a polynucleotide according to claim 20. 22. A hybridoma cell for the production of a monoclonal antibody that specifically binds to the human poliovirus receptor (PVR), according to any one of claims. 1-15, wherein the hybridoma cell comprises a polynucleotide according to claim 18 and a polynucleotide according to claim 20. 23. A pharmaceutical composition for modulating the immune system by inhibiting the binding of PVR to TIGIT, containing as an active ingredient an effective amount of at least one isolated antibody or an antibody fragment thereof containing at least an antigen-binding part, according to any one of claims. 1-15, and a pharmaceutically acceptable excipient, diluent, salt or carrier. 24. A pharmaceutical composition according to claim 23 for use in the treatment of cancer. 25. A method of treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition according to claim 23. 26. The method of claim 25, further comprising administering to said subject an additional immunomodulator, an activated lymphocyte, a kinase inhibitor, a chemotherapeutic agent, or any other anti-cancer agent. 27. The method of claim 25, wherein the additional immunomodulator is an antibody to an immune checkpoint molecule selected from the group consisting of PD-1, CTLA-4, PD-L1, CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activation gene 3 (LAG3), CD137, OX40 (also called CD134), killer cell immunoglobulin-like receptors (KIR), TIGIT, and any combination thereof. 28. The method of claim 25, wherein the cancer is solid cancer. 29. The method of claim 25, wherein the cancer is hematologic cancer. 30. The method of claim 25, wherein the treatment results in the prevention or reduction of the formation, growth, or spread of metastases in the subject. 31. A method for diagnosing cancer in a subject, the method comprising bringing a biological sample into contact with an antibody or an antibody fragment according to any one of claims. 1-15, determining the expression or activity of PVR in a sample from a subject, and comparing the expression or activity of the PVR with a reference value for the expression or activity of PVR.

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